Amyotrophic Lateral Sclerosis Transgenic Mice Research Articles

FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model

Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative diseases, yet effective treatment is lacking. Moreover, the underlying pathomechanisms of ALS remain unclear, with impaired mitophagy function being increasingly recognized as a contributing factor. FUN14 domain-containing protein 1 (FUNDC1) is an autophagy receptor localized to the outer mitochondrial membrane and a mitochondrial membrane protein that mediates mitophagy and therefore considered as important factor in neurodegenerative diseases. However, its specific role in ALS is not yet clear. Therefore, this study aimed to investigate the regulatory role of FUNDC1 in ALS and determine its regulatory mechanisms. ALS transgenic mice were obtained and maintained under standard conditions. Cell lines were generated by stable transfection with hSOD1G93A or control vectors. Mice received intrathecal injections of AAV9 vectors expressing FUNDC1 or EGFP. Motor function was assessed through behavioral tests, and histological and immunostaining analyses were performed. Colocalization analysis was conducted in transfected cells, and protein expression was evaluated via western blotting. We first observed that FUNDC1 was significantly downregulated in the spinal cord tissues of SOD1G93A mice. FUNDC1 overexpression considerably improved locomotor activity and prolonged survival time in SOD1G93A mice. Mechanistically, reduced expression of FUNDC1 resulted in decreased mitophagy, as indicated by decreased recruitment through LC3 in SOD1G93A mice and cellular models. Consequently, this led to increased mitochondrial accumulation and cell apoptosis, exacerbating the ALS phenotype. Furthermore, we identified transcription factor FOXD3 as an essential upstream factor of FUNDC1, resulting in reduced transcription of FUNDC1 in ALS lesions. This study suggests a novel strategy of targeting FUNDC1-mediated mitophagy for developing therapeutic interventions to mitigate disease progression and improve outcomes for ALS patients.

SERCA1 Overexpression in Skeletal Muscle Attenuates Muscle Atrophy and Improves Motor Function in a Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of muscle mass and muscle function. Previous work from our lab demonstrated that skeletal muscles from a mouse model of ALS show elevated intracellular calcium (Ca2+) levels and heightened endoplasmic reticulum (ER) stress. To investigate whether overexpression of sarcoplasmic reticulum (SR) Ca2+ ATPase 1 (SERCA1) in skeletal muscle would improve intracellular Ca2+ handling, attenuate ER stress, and improve motor function ALS transgenic mice. B6SJL-Tg (SOD1*G93A)1Gur/J (ALS-Tg) mice were bred with skeletal muscle α-actinin SERCA1 overexpressing mice to generate wild type (WT), SERCA1 overexpression (WT/+SERCA1), ALS-Tg, and SERCA1 overexpressing ALS-Tg (ALS-Tg/+SERCA1) mice. Motor function (grip test) was assessed weekly and skeletal muscles were harvested at 16 weeks of age to evaluate muscle mass, SR-Ca2+ ATPase activity, levels of SERCA1 and ER stress proteins - protein disulfide isomerase (PDI), Grp78/BiP, and C/EBP homologous protein (CHOP). Single muscle fibers were also isolated from the flexor digitorum brevis muscle to assess changes in resting and peak Fura-2 ratios. ALS-Tg/+SERCA1 mice showed improved motor function, delayed onset of disease, and improved muscle mass compared to ALS-Tg. Further, ALS-Tg/+SERCA1 mice returned levels of SERCA1 protein and SR-Ca2+ ATPase activity back to levels in WT mice. Unexpectedly, SERCA-1 overexpression increased levels of the ER stress maker Grp78/BiP in both WT and ALS-Tg mice, while not altering protein levels of PDI or CHOP. Lastly, single muscle fibers from ALS-Tg/+SERCA1 had similar resting but lower peak Fura-2 levels (at 30 Hz and 100 Hz) compared to ALS-Tg mice. These data indicate that SERCA1 overexpression attenuates the progressive loss of muscle mass and maintains motor function in ALS-Tg mice while not lowering resting Ca2+ levels or ER stress.

SPY1 inhibits neuronal ferroptosis in amyotrophic lateral sclerosis by reducing lipid peroxidation through regulation of GCH1 and TFR1

Ferroptosis is an iron-dependent cell death with the accumulation of lipid peroxidation and dysfunction of antioxidant systems. As the critical regulator, glutathione peroxidase 4 (GPX4) has been demonstrated to be down-regulated in amyotrophic lateral sclerosis (ALS). However, the mechanism of ferroptosis in ALS remains unclear. In this research, bioinformatics analysis revealed a high correlation between ALS, ferroptosis, and Speedy/RINGO cell cycle regulator family member A (SPY1). Lipid peroxidation of ferroptosis in hSOD1G93A cells and mice was generated by TFR1-imported excess free iron, decreased GSH, mitochondrial membrane dysfunction, upregulated ALOX15, and inactivation of GCH1, GPX4. SPY1 is a “cyclin-like” protein that has been proved to enhance the viability of hSOD1G93A cells by inhibiting DNA damage. In our study, the decreased expression of SPY1 in ALS was resulted from unprecedented ubiquitination degradation mediated by MDM2 (a nuclear-localized E3 ubiquitin ligase). Further, SPY1 was identified as a novel ferroptosis suppressor via alleviating lipid peroxidation produced by dysregulated GCH1/BH4 axis (a resistance axis of ferroptosis) and transferrin receptor protein 1 (TFR1)-induced iron. Additionally, neuron-specific overexpression of SPY1 significantly delayed the occurrence and prolonged the survival in ALS transgenic mice through the above two pathways. These results suggest that SPY1 is a novel target for both ferroptosis and ALS.

The mechanism of the WNT5A and FZD4 receptor mediated WNT/β–catenin pathway in the degeneration of ALS spinal cord motor neurons

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with unknown etiology, characterized by motor neuron degeneration, and there is no highly effective treatment. The canonical WNT/β-catenin signaling pathway has a critical role in the physiological and pathophysiological processes of the central nervous system. In this study, we investigated the regulatory mechanism of the WNT/β-catenin signaling pathway from the perspective of ligand-receptor binding and its relationship with the degeneration of ALS motor neurons. We used hSOD1-G93A mutant ALS transgenic mice and hSOD1-G93A mutant NSC34 cells combined with morphological and molecular biology techniques to determine the role of the WNT/β–catenin pathway in ALS. Our findings demonstrated that WNT5A regulates the WNT/β-catenin signaling pathway by binding to the FZD4 receptor in the pathogenesis of ALS and affects the proliferation and apoptosis of ALS motor neurons. Therefore, these findings may lead to the development of novel therapies to support the survival of ALS motor neurons.

Targeting the epigenome to treat neurodegenerative diseases or delay their onset: a perspective

Targeting the epigenome to treat neurodegenerative diseases or delay their onset: a perspective

Perturbations of the Proteome and of Secreted Metabolites in Primary Astrocytes from the hSOD1(G93A) ALS Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins—and of secreted metabolites—involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome–lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte–MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis.

Microglia Morphological Changes in the Motor Cortex of hSOD1G93A Transgenic ALS Mice.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of spinal motor neurons as well as corticospinal (CSN) large pyramidal neurons within cortex layer V. An intense microglia immune response has been associated with both upper and lower motor neuron degeneration in ALS patients, whereas microgliosis occurrence in the motor cortex of hSOD1G93A mice—the best characterized model of this disease—is not clear and remains under debate. Since the impact of microglia cells in the neuronal environment seems to be crucial for both the initiation and the progression of the disease, here we analyzed the motor cortex of hSOD1G93A mice at the onset of symptoms by the immunolabeling of Iba1/TMEM119 double positive cells and confocal microscopy. By means of Sholl analysis, we were able to identify and quantify the presence of presumably activated Iba1/TMEM119-positive microglia cells with shorter and thicker processes as compared to the normal surveilling and more ramified microglia present in WT cortices. We strongly believe that being able to analyze microglia activation in the motor cortex of hSOD1G93A mice is of great importance for defining the timing and the extent of microglia involvement in CSN degeneration and for the identification of the initiation stages of this disease.

Effect of electroacupuncture on expression of heat shock protein 70 in mice with amyotrophic lateral sclerosis

To observe the effects of electroacupuncture (EA) on the body weight, disease progression and the expression of heat shock protein 70 (HSP70) in lumbar spinal cord of amyotrophic lateral sclerosis (ALS) mice, so as to explore the mechanism of EA on treating ALS. Eighteen ALS transgenic SOD1G93A mice were randomly divided into model, EA and medication groups, with 6 mice in each group, and six C57BL/6J mice were used as the normal group. Mice in the EA group received EA at "Quchi"(LI11)-"Hegu"(LI4), "Zusanli"(ST36)- "Sanyinjiao"(SP6), 30 min/time, 5 times/week, for 8 weeks.Mice in the medication group were given riluzole solution (7.6 mg·kg-1·d-1) by gavage for 8 weeks. The body weight of the mice was recorded and the motor function of the hind limbs was evaluated by the neurological function scoring stan-dard of the ALS Therapeutic Development Institute. The expression of HSP70 in lumbar spinal cord was detected by Western blot and immunohistochemistry, respectively. Compared with the normal group, the body weight and the expression of HSP70 in the model group decreased significantly (P<0.05), while no significant difference in the body weight was found among other groups(P>0.05). After intervention and in comparison with the model group, the disease onset time and paralysis time of the EA group and the medication group were significantly delayed (P<0.05, P<0.01), the expression of HSP70 in the EA group and the medicine group was significantly increased (P<0.05, P<0.01).But there was no significant difference in the survival time among all groups(P＞0.05). The disease onset time of the EA group was shorter than that in the medication group (P<0.05). EA can increase the expression of HSP70 in lumbar spinal cord, thereby delaying the progression of ALS.

Potential Roles of the WNT Signaling Pathway in Amyotrophic Lateral Sclerosis.

The WNT signaling pathway plays an important role in the physiological and pathophysiological processes of the central nervous system and the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We reviewed the literature pertinent to WNT/β–catenin signaling in ALS from cellular studies, animal models, and human clinical trials. WNT, WNT receptors, and other components of the WNT signaling pathway are expressed in both ALS patients and transgenic mice, and are involved in the pathogenesis of ALS. Studies have shown that abnormal activation of the WNT/β–catenin signaling pathway is related to neuronal degeneration and glial cell proliferation. WNT/Ca2+ signaling is associated with the pro–inflammatory phenotype of microglia; data on the muscle skeletal receptor Tyr kinase receptor in superoxide dismutase–1–G93A mice indicate that gene therapy is necessary for successful treatment of ALS. The varying profiles of lipoprotein receptor–related protein 4 antibodies in different ethnic groups suggest that individual treatment and multifactorial personalized approaches may be necessary for effective ALS therapy. In conclusion, the WNT signaling pathway is important to the ALS disease process, making it a likely therapeutic target.

Satellite Glial Cells of the Dorsal Root Ganglion: A New "Guest/Physiopathological Target" in ALS.

Introduction: Amyotrophic lateral sclerosis (ALS) might not only be circumscribed to the motor system but also involves other neuronal systems including sensory abnormalities. In line with this notion, we aimed to assess the pathophysiology of sensory disturbances in the SOD1G93A mouse model of ALS, focusing on the satellite glial cells (SGCs) at the dorsal root ganglion (DRG) as a new potential target of the disease.Material and Methods: The presence of sensory disturbances was evaluated using von Frey, hot plate, and hot water tail immersion tests at 75 days old, which represented the motor-pre-symptomatic stage. Cell biology analysis was performed at 75 and 95 days old and included conventional histology, immunofluorescence, and electron microscopy of sensory neuron-SGC unit dissociates as a well as western blotting from DRG lysates.Results: At 75 days old, von Frey and hot plate tests demonstrated clear thermoalgesic disturbances in ALS transgenic mice. Histological studies of the SN-SGC units revealed abnormal SOD1 accumulation, which was associated with nitro-oxidative stress and biogenesis of lipid droplets in SGCs. Interestingly, these alterations led to a progressive lysosomal storage disorder and occasionally vacuolar degeneration in SGCs.Conclusions: SGCs emerge as a primary pathophysiological target in the SOD1 transgenic murine model of ALS, clearly reinforcing the pathogenic role of glial cells in motor neuron disease. Presymptomatic alterations of SGCs, might not only be responsible of sensory disturbances in ALS, but due to spinal cord sensory-motor circuits could also contribute to anterior horn motor disturbances.

Novel Selenium-based compounds with therapeutic potential for SOD1-linked amyotrophic lateral sclerosis.

BackgroundAmyotrophic lateral sclerosis (ALS), also known as motor neuron disease as well as Lou Gehrig's disease, is a progressive neurological disorder selectively affecting motor neurons with no currently known cure. Around 20% of the familial ALS cases arise from dominant mutations in the sod1 gene encoding superoxide dismutase1 (SOD1) enzyme. Aggregation of mutant SOD1 in familial cases and of wild-type SOD1 in at least some sporadic ALS cases is one of the known causes of the disease. Riluzole, approved in 1995 and edaravone in 2017 remain the only drugs with limited therapeutic benefits.MethodsWe have utilised the ebselen template to develop novel compounds that redeem stability of mutant SOD1 dimer and prevent aggregation. Binding modes of compounds have been visualised by crystallography. In vitro neuroprotection and toxicity of lead compounds have been performed in mouse neuronal cells and disease onset delay of ebselen has been demonstrated in transgenic ALS mice model.FindingWe have developed a number of ebselen-based compounds with improvements in A4V SOD1 stabilisation and in vitro therapeutic effects with significantly better potency than edaravone. Structure-activity relationship of hits has been guided by high resolution structures of ligand-bound A4V SOD1. We also show clear disease onset delay of ebselen in transgenic ALS mice model holding encouraging promise for potential therapeutic compounds.InterpretationOur finding established the new generation of organo-selenium compounds with better in vitro neuroprotective activity than edaravone. The potential of this class of compounds may offer an alternative therapeutic agent for ALS treatment. The ability of these compounds to target cysteine 111 in SOD may have wider therapeutic applications targeting cysteines of enzymes involved in pathogenic and viral diseases including main protease of SARS-Cov-2 (COVID-19).FundingProject funding was supported by the 10.13039/100000971ALS Association grant (WA1128) and Fostering Joint International Research (19KK0214) from the 10.13039/100009950Ministry of Education, Culture, Sports, Science and Technology (10.13039/501100001700MEXT), Japan.

Absence of Receptor for Advanced Glycation End Product (RAGE) Reduces Inflammation and Extends Survival in the hSOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron degenerative disease that is without effective treatment. The receptor for advanced glycation end products (RAGE) is a major component of the innate immune system that has been implicated in ALS pathogenesis. However, the contribution of RAGE signalling to the neuroinflammation that underlies ALS neurodegeneration remains unknown. The present study therefore generated SOD1G93A mice lacking RAGE and compared them with SOD1G93A transgenic ALS mice in respect to disease progression (i.e. body weight, survival and muscle strength), neuroinflammation and denervation markers in the spinal cord and tibialis anterior muscle. We found that complete absence of RAGE signalling exerted a protective effect on SOD1G93A pathology, slowing disease progression and significantly extending survival by ~ 3weeks and improving motor function (rotarod and grip strength). This was associated with reduced microgliosis, cytokines, innate immune factors (complement, TLRs, inflammasomes), and oxidative stress in the spinal cord, and a reduction of denervation markers in the tibialis anterior muscle. We also documented that RAGE mRNA expression was significantly increased in the spinal cord and muscles of preclinical SOD1 and TDP43 models of ALS, supporting a widespread involvement for RAGE in ALS pathology. In summary, our results indicate that RAGE signalling drives neuroinflammation and contributes to neurodegeneration in ALS and highlights RAGE as a potential immune therapeutic target for ALS.

The deletion of mutant SOD1 via CRISPR/Cas9/sgRNA prolongs survival in an amyotrophic lateral sclerosis mouse model.

The superoxide dismutase 1 (SOD1) mutation is one of the most notable causes of amyotrophic lateral sclerosis (ALS), and modifying the mutant SOD1 gene is the best approach for the treatment of patients with ALS linked to the mutations in this gene. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas9)/sgRNA delivered by the adeno-associated virus (AAV) system is a powerful tool for genome editing in the central nervous system (CNS). Here, we tested the capacity of the AAV-SaCas9-sgRNA system to modify mutant SOD1 in SOD1G93A transgenic mice and found that AAV9-SaCas9-sgRNA5 deleted the SOD1 gene, improved the lifespan of SOD1G93A mice by 54.6%, and notably ameliorated the performance of ALS transgenic mice. An immunochemical analysis showed that the expression of mutant SOD1 was very weak in motor neurons expressing SaCas9-sgRNA5. Consequently, the area showing muscle atrophy was more notably restored in the group treated with SaCas9-sgRNA5 compared with the group treated with SaCas9-sgLacZ. In addition, deep sequencing did not show the indel mutation in the gene highly matched to sgRNA5. Hence, AAV9-SaCas9-sgRNA-based gene editing is a feasible potential treatment for patients with ALS linked to SOD1 mutations.

β-catenin aggregation in models of ALS motor neurons: GSK3β inhibition effect and neuronal differentiation

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. A 20% of familial ALS cases are associated with mutations in the gene coding for superoxide dismutase 1 (SOD1). The accumulation of abnormal aggregates of different proteins is a common feature in motor neurons of patients and transgenic ALS mice models, which are thought to contribute to disease pathogenesis. Developmental morphogens, such as the Wnt family, regulate numerous features of neuronal physiology in the adult brain and have been implicated in neurodegeneration. β-catenin is a central mediator of both, Wnt signaling activity and cell-cell interactions. We previously reported that the expression of mutant SOD1 in the NSC34 motor neuron cell line decreases basal Wnt pathway activity, which correlates with cytosolic β-catenin accumulation and impaired neuronal differentiation. In this work, we aimed a deeper characterization of β-catenin distribution in models of ALS motor neurons. We observed extensive accumulation of β-catenin supramolecular structures in motor neuron somas of pre-symptomatic mutant SOD1 mice. In cell-cell appositional zones of NSC34 cells expressing mutant SOD1, β-catenin displays a reduced co-distribution with E-cadherin accompanied by an increased association with the gap junction protein Connexin-43; these findings correlate with impaired intercellular adhesion and exacerbated cell coupling. Remarkably, pharmacological inhibition of the glycogen synthase kinase-3β (GSK3β) in both NSC34 cell lines reverted both, β-catenin aggregation and the adverse effects of mutant SOD1 expression on neuronal differentiation. Our findings suggest that early defects in β-catenin distribution could be an underlying factor affecting the onset of neurodegeneration in familial ALS.

Deletion of Tbk1 disrupts autophagy and reproduces behavioral and locomotor symptoms of FTD-ALS in mice

Haploinsufficiency of the protein kinase Tbk1 has shown to cause both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, the pathogenic mechanisms are unclear. Here we show that conditional neuronal deletion of Tbk1 in leads to cognitive and locomotor deficits in mice. Tbk1-NKO mice exhibited numerous neuropathological changes, including neurofibrillary tangles, abnormal dendrites, reduced dendritic spine density, and cortical synapse loss. The Purkinje cell layer of the cerebellum presented dendritic swelling, abnormally shaped astrocytes, and p62- and ubiquitin-positive aggregates, suggesting impaired autophagy. Inhibition of autophagic flux with bafilomycin A increased total Tkb1 levels in motor neuron-like cells in vitro, suggesting autophagy-dependent degradation of Tbk1. Although Tbk1 over-expression did not affect mutant SOD1 levels in SOD1G93A-transfected cells, it increased the soluble/insoluble ratio and reduced the number and size of SOD1G93A aggregates. Finally, in vivo experiments showed that Tkb1 expression was reduced in SOD1G93A ALS transgenic mice, which showed decreased p62 protein aggregation and extended survival after ICV injection of adeno-associated viral vectors encoding Tbk1. These data shed light on the neuropathological changes that result from Tbk1 deficiency and hint at impaired autophagy as a contributing factor to the cognitive and locomotor deficits that characterize FTD-ALS in patients with Tkb1 haploinsufficiency.

Analysis of the therapeutic potential of different administration routes and frequencies of human mesenchymal stromal cells in the SOD1G93A mouse model of amyotrophic lateral sclerosis.

Cellular therapy represents a novel option for the treatment of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Its major aim is the generation of a protective environment for degenerating motor neurons. Mesenchymal stromal cells secrete different growth factors and have antiapoptotic and immunomodulatory properties. They can easily and safely be isolated from human bone marrow and are therefore considered promising therapeutic candidates. In the present study, we compared intraventricular application of human mesenchymal stromal cells (hMSCs) versus single and repeated intraspinal injections in the mutant SOD1G93A transgenic ALS mouse model. We observed significant reduction of lifespan of animals treated by intraventricular hMSC injection compared with the vehicle treated control group, accompanied by changes in weight, general condition, and behavioural assessments. A potential explanation for these rather surprising deleterious effects lies in increased microgliosis detected in the hMSC treated animals. Repeated intraspinal injection at two time points resulted in a slight but not significant increase in survival and significant improvement of motor performance although no hMSC-induced changes of motor neuron numbers, astrogliosis, and microgliosis were detected. Quantitative real time polymerase chain reaction showed reduced expression of endothelial growth factor in animals having received hMSCs twice compared with the vehicle treated control group. hMSCs were detectable at the injection site at Day 20 after injection into the spinal cord but no longer at Day 70. Intraspinal injection of hMSCs may therefore be a more promising option for the treatment of ALS than intraventricular injection and repeated injections might be necessary to obtain substantial therapeutic benefit.

The role of insulin-like growth factor 1 in ALS cell and mouse models: A mitochondrial protector

Amyotrophic lateral sclerosis (ALS) is a common neurodegenerative disorder, but little is known about the exact causes and pathophysiology of this disease. In transgenic mouse models of ALS, mitochondrial abnormalities develop during the disease and might contribute to the progression of ALS. Gene therapy was recently shown to induce beneficial effects. For example, the delivery of human insulin-like growth factor-1 (hIGF-1) by self-complementary adeno-associated virus (AAV) vectors has been shown to prolong the lifespan of ALS transgenic mice. However, the function of IGF-1 in mitochondria has not been systematically studied in ALS models. In this study, scAAV9-hIGF-1 was intramuscularly injected into transgenic SOD1G93A mice and administered to cell lines expressing the ∼25-kDa C-terminal fragment of transactive response DNA-binding protein (TDP-25). The mitochondrial electrical transmembrane potential was hyperpolarized, and electron microscopy findings revealed that the abnormal mitochondria were transformed. Moreover, the intrinsic mitochondrial apoptotic process was modified through the upregulation of anti-apoptotic proteins (B-cell lymphoma-extra large (Bcl-xl) and B-cell lymphoma-2 (Bcl-2)), the downregulation of pro-apoptotic proteins (Bcl-2-associated x protein (Bax) and Bcl-2 homologous antagonist killer (Bak)) and a reduction in mitochondrial cytochrome c release. Mitophagy was also increased after scAAV9-hIGF-1 treatment, as evidenced by a decrease in the p62 level and an increase in the LC3-II level. Furthermore, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system was used to delete the IGF-1 gene in SOD1G93A model mice via an intrathecal injection of scAAV9-sgRNA-IGF1-Cas9 to confirm these findings. The protective effect of IGF-1 on the mitochondria decreased after genetic deletion. These novel findings demonstrate that IGF-1 strongly protects mitochondria from apoptosis and upregulates mitophagy in mouse and cell models of ALS. Therefore, therapies that specifically protect mitochondrial function might be promising strategies for treating ALS.

Adipose-derived stem cell conditioned medium impacts asymptomatic peripheral neuromuscular denervation in the mutant superoxide dismutase (G93A) transgenic mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is devastating, leading to paralysis and death. Disease onset begins pre-symptomatically through spinal motor neuron (MN) axon die-back from musculature at ∼47 days of age in the mutant superoxide dismutase 1 (mSOD1G93A) transgenic ALS mouse model. This period may be optimal to assess potential therapies. We previously demonstrated that post-symptomatic adipose-derived stem cell conditioned medium (ASC-CM) treatment is neuroprotective in mSOD1G93A mice. We hypothesized that early disease onset treatment could ameliorate neuromuscular junction (NMJ) disruption. To determine whether pre-symptom administration of ASC-CM prevents early NMJ disconnection. We confirmed the NMJ denervation time course in mSOD1G93A mice using co-labeling of neurofilament and post-synaptic acetylcholine receptors (AchR) by α-bungarotoxin. We determined whether ASC-CM ameliorates early NMJ loss in mSOD1G93A mice by systemically administering 200μl ASC-CM or vehicle medium daily from post-natal days 35 to 47 and quantifying intact NMJs through co-labeling of neurofilament and synaptophysin with α-bungarotoxin in gastrocnemius muscle. Intact NMJs were significantly decreased in 47 day old mSOD1G93A mice (p < 0.05), and daily systemic ASC-CM prevented disease-induced NMJ denervation compared to vehicle treated mice (p < 0.05). Our results lay the foundation for testing the long-term neurological benefits of systemic ASC-CM therapy in the mSOD1G93A mouse model of ALS.

ScAAV9-VEGF-165 inhibits neuroinflammatory responses and invasion of macrophages into the peripheral nervous system of ALS transgenic mice

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that leads to paralysis and death within 3–5 years. Although the vast majority of studies have focused on vulnerable neurons, growing evidence has shown that non-neuronal cells contribute to the pathogenesis and disease progression. Here, we showed that intrathecal injection of scAAV9-VEGF at 60 days of age significantly reduced the number of microglia and inhibited the neuroinflammatory response in the CNS. Meanwhile, we found that administration of VEGF inhibited the invasion of macrophages into the PNS, including ventral nerve roots, sciatic nerves and muscles. Overall, our study indicated the anti-inflammation effect of VEGF in the CNS and PNS of ALS mice when delivered by intrathecal injection.

The D2 Dopamine Receptor Interferes With the Protective Effect of the A2A Adenosine Receptor on TDP-43 Mislocalization in Experimental Models of Motor Neuron Degeneration.

The A2A adenosine receptor (A2AR) and D2 dopamine receptor (D2R) are two G-protein-coupled receptors that can form dimers and negatively regulate their partners. TAR DNA-binding protein (TDP-43) is a nuclear protein that has been implicated in amyotrophic lateral sclerosis (ALS). Mislocalization of TDP-43 from the nucleus to the cytoplasm is an early step of TDP-43 proteinopathy. Our previous studies indicated that A2AR is a potential drug target for ALS because treatment with an A2AR agonist (JMF1907; a T1-11 analog) prevents reactive oxygen species (ROS)-induced TDP-43 mislocalization in a motor neuron cell line (NSC34) and delays motor impairment in a TDP-43 transgenic ALS mouse model. Here, we set out to assess whether activation of D2R interferes with the beneficial effects of an A2AR agonist on motor neurons. We first demonstrated that A2AR and D2R are both located in motor neurons of mouse and human spinal cords and human iPSC-derived motor neurons. Expression of A2AR and D2R in NSC34 cells led to dimer formation without affecting the binding affinity of A2AR toward T1-11. Importantly, activation of D2R reduced T1-11-mediated activation of cAMP/PKA signaling and subsequent inhibition of TDP-43 mislocalization in NSC34 cells. Treatment with quinpirole (a D2 agonist) blunted the rescuing effect of T1-11 on TDP-43 mislocalization and impaired grip strength in a mouse model of ALS. Our findings suggest that D2R activation may limit the beneficial responses of an A2AR agonist in motor neurons and may have an important role in ALS pathogenesis.