Intrathecal application of neuroectodermally converted stem cells into a mouse model of ALS: limited intraparenchymal migration and survival narrows therapeutic effects

Summary Stem and progenitor cells provide a promising therapeutic strategy for amyotrophic lateral sclerosis (ALS). To comparatively evaluate the therapeutic potentials of human bone marrow-derived mesodermal stromal cells (hMSCs) and umbilical cord blood cells (hUBCs) in ALS, we transplanted hMSCs and hUBCs and their neuroectodermal derivatives (hMSC-NSCs and hUBC-NSCs) into the ALS mouse model over-expressing the G93A mutant of the human SOD1 gene. We used a standardized protocol similar to clinical studies by performing a power calculation to estimate sample size prior to transplantation, matching the treatment groups for gender and hSOD-G93A gene content, and applying a novel method for directly injecting 100,000 cells into the CSF (the cisterna magna). Ten days after transplantation we found many cells within the subarachnoidal space ranging from frontal basal cisterns back to the cisterna magna, but only a few cells around the spinal cord. hMSCs and hMSC-NSCs were also located within the Purkinje cell layer. Intrathecal cell application did not affect survival times of mice compared to controls. Consistently, time of disease onset and first pareses, death weight, and motor neuron count in lumbar spinal cord did not vary between treatment groups. Interestingly, transplantation of hMSCs led to an increase of pre-symptomatic motor performance compared to controls in female animals. The negative outcome of the present study is most likely due to insufficient cell numbers within the affected brain regions (mainly the spinal cord). Further experiments defining the optimal cell dose, time point and route of application and particularly strategies to improve the homing of transplanted cells towards the CNS region of interest are warranted to define the therapeutic potential of mesodermal stem cells for the treatment of ALS.     

小胶质细胞在SOD1-G93A转基因小鼠腰髓中的变化

目的观察小胶质细胞在SOD1-G93A转基因小鼠不同时期腰髓中的变化,探讨小胶质细胞活化与肌萎缩侧索硬化(ALS)疾病进展的关系。方法以国际公认的SOD1-G93A转基因小鼠,应用免疫组化、激光共聚焦显微镜及Westernblot方法 ,分别观察SOD1-G93A转基因小鼠症状前期、症状期、终末期及其同窝对照腰髓小胶质细胞形态数量及特异性标记物表达的变化情况。结果 SOD1-G93A转基因小鼠腰髓在症状前期(60天)已出现小胶质细胞数量增多及特异性标记物CD11b表达升高,随病程进展,症状期小胶质细胞增多、活化显著,终末期达高峰。结论随SOD1-G93A转基因小鼠病程进展小胶质细胞增生明显,小胶质细胞的活化可能参与ALS运动神经元损伤。     

Relationship between neuropathology and disease progression in the SOD1(G93A) ALS mouse

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons. However, recent reports suggest an active role of non-neuronal cells in the pathogenesis of the disease. Here, we examined quantitatively the temporal development of neuropathologic features in the brain and spinal cord of a mouse model of ALS (SOD1^G93A). Four phases of the disease were studied in both male and female SOD1^G93A mice: presymptomatic (PRE-SYM), symptomatic (SYM), endstage (ES) and moribund (MB). Compared to their control littermates, SOD1^G93A mice showed an increase in astrogliosis in the motor cortex, spinal cord and motor trigeminal nucleus in the SYM phase that worsened progressively in ES and MB animals. Associated with this increase in astrogliosis was a concomitant increase in motor neuron cell death in the spinal cord and motor trigeminal nucleus in both ES and MB mice, as well as in the ventrolateral thalamus in MB animals. In contrast, microglial activation was significantly increased in all the same regions but only when the mice were in the MB phase. These results suggest that astrogliosis preceded or occurred concurrently with neuronal degeneration whereas prominent microgliosis was evident later (MB stage), after significant motor neuron degeneration had occurred. Hence, our findings support a role for astrocytes in modulating the progression of non-cell autonomous degeneration of motor neurons, with microglia playing a role in clearing degenerating neurons.     

Appearance of phagocytic microglia adjacent to motoneurons in spinal cord tissue from a presymptomatic transgenic rat model of amyotrophic lateral sclerosis

Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu²⁺/Zn²⁺ superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1^H46R transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor‐α (TNFα) and monocyte chemoattractant protein‐1 (MCP‐1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage. © 2010 Wiley‐Liss, Inc.     

Immune reactivity in a mouse model of familial ALS correlates with disease progression

OBJECTIVE: The cause of motor neuron death in ALS is incompletely understood. This study aims to define the potential involvement of nonneuronal immune-inflammatory factors in the destruction of motor neurons in mutant superoxide dismutase-1 (SOD1) transgenic mice as a model of ALS. BACKGROUND: The presence of activated microglia, IgG and its receptor for Fc portion (FcgammaRI), and T lymphocytes in the spinal cord of both patients with ALS and experimental animal models of motor neuron disease strongly suggests that immune-inflammatory factors may be actively involved in the disease process. METHODS: The expression of immune-inflammatory factors was followed in both human mutant (G93A) SOD1 transgenic mice and human wild-type SOD1 transgenic mice, at different ages (40, 80, and 120 days). Fixed, frozen, free-floating sections of the lumbar spinal cord were stained with antibodies against CD11b, IgG, FcgammaRI, intercellular adhesion molecule-1 (ICAM-1), CD3, and glial fibrillary acidic protein. RESULTS: The earliest change observed was the upregulation of ICAM-1 in the ventral lumbar spinal cord of 40-day-old mutant SOD1 mice. IgG and FcgammaRI reactivities were detected on motor neurons as early as 40 days and on microglial cells at later stages. Microglial activation was first evident in the ventral horn at 80 days, whereas reactive astrocytes and T cells became most prominent in 120-day-old mutant SOD1 mice. CONCLUSION: The upregulation of proinflammatory factors during early presymptomatic stages as well as the expansion of immune activation as disease progresses in mutant SOD1 transgenic mice suggest that immune-inflammatory mechanisms could contribute to disease progression.     

人骨髓间质干细胞经心脏移植治疗超氧化物歧化酶1-G93A转基因鼠的疗效

目的 研究经心脏移植入骨髓间质干细胞(hMSCs)对肌萎缩侧索硬化(ALS)模型鼠发病时间、生存期和病理的影响.方法 体外培养扩增hMSCs,流式细胞仪鉴定其性质及纯度.将3×10~6个第5代hMSCs经心脏移植入预放疗的8周龄超氧化物歧化酶1(SOD1)-G93A转基因鼠,用Weyd4分法评定移植鼠和未治疗鼠的生存期、发病时间,采取尼氏染色计数脊髓前角运动神经元,通过免疫荧光检测人特异性核抗原验证hMSCs在受体鼠中枢神经系统中的植入.结果 生存分析显示,经心脏移植hMSCs的ALS模型鼠平均发病时间为(172.85±3.82)d,比未治疗组[(156.56±3.60)d]延迟16 d,差异有统计学意义(x~2=10.888,P=0.001);hMSCs经心脏移植组平均生存期为(202.19±4.09)d,比未治疗组[(188.32±3.51)d]延长14 d,差异有统计学意义(x~2=3.917,P=0.04).尼氏染色显示在20周时移植鼠脊髓前角大运动神经元计数多于未治疗鼠;终末期hMSCs移植鼠中,在脑和脊髓前角病变区可检测到人特异性核抗原.结论 hMSCs可经心脏移植,在ALS模型鼠中可长期植入,延长生存期,延缓脊髓前角运动神经元的丢失.     

Treatment with edaravone, initiated at symptom onset, slows motor decline and decreases SOD1 deposition in ALS mice

Edaravone is a free-radical scavenger, an agent being widely used for cerebral ischemia in Japan. To evaluate its efficacy for possible treatment of amyotrophic lateral sclerosis (ALS), we performed a randomized blind trial in ALS model mice. After identification of the clinical onset in each female G93A mutant SOD1 transgenic mouse, we intraperitoneally administered multiple doses of edaravone to the mice and observed their motor symptoms. We also counted the number of lumbar motoneurons, determined the 3-nitrotyrosine/tyrosine ratio, and evaluated the abnormal SOD1 aggregation in the spinal cord at the 10th day after the edaravone injection. Edaravone significantly slowed the motor decline of the transgenic mice. The remaining motoneurons were significantly preserved in the higher-dose edaravone-administered group, and the 3-nitrotyrosine/tyrosine ratios were reduced dose-dependently. Intriguingly, the area of abnormal SOD1 deposition in the spinal cord was significantly decreased in the higher-dose edaravone-administered group. Our results indicate that edaravone was effective to slow symptom progression and motor neuron degeneration in the ALS model mice. These favorable actions might be attributable to the yet unidentified mechanism responsible for reducing the deposition of mutant SOD1.     

Acute glial activation by stab injuries does not lead to overt damage or motor neuron degeneration in the G93A mutant SOD1 rat model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons within the brain and spinal cord are lost, leading to paralysis and death. Recently, a correlation between head trauma and the incidence of ALS has been reported. Furthermore, new invasive neurosurgical studies are being planned which involve inserting needles directly to the spinal cord. We therefore tested whether acute trauma to the spinal cord via a knife wound injury would lead to accelerated disease progression in rodent models of ALS (SOD1^G93A rats). A longitudinal stab injury using a small knife was performed within the lumbar spinal cord region of presymptomatic SOD1^G93A rats. Host glial activation was detected in the lumbar area surrounding a micro-knife lesion at 2 weeks after surgery in both wild type and SOD1^G93A animals. However, there was no sign of motor neuron loss in the injured spinal cord of any animal and normal motor function was maintained in the ipsilateral limb. These results indicate that motor neurons in presymptomatic G93A animals are not affected by an invasive puncture wound injury involving reactive astrocytes. Furthermore, acute trauma alone does not accelerate disease onset or progression in this ALS model which is important for future strategies of gene and cell therapies directly targeting the spinal cord of ALS patients.     

NLRP3 inflammasome is expressed by astrocytes in the SOD1 mouse model of ALS and in human sporadic ALS patients

Amyotrophic lateral sclerosis (ALS) is characterized by the degeneration of motoneurons in the cerebral cortex, brainstem and spinal cord. Neuroinflammation plays an important role in the pathogenesis of ALS and involves the activation of microglia and astrocytes. Intracellular inflammasome complexes are part of the innate immunity as they sense and execute host inflammatory responses. The best characterized component is the NLRP3 inflammasome comprised of the NLR protein NLRP3, the adaptor ASC and pro‐caspase 1. The NLRP3 inflammasome is critical for the activation of caspase 1 and the processing and release of IL1β and IL18. In this study, we investigated the expression, activation and co‐localization of the NLRP3 inflammasome in the spinal cord of male SOD1(G93A) mice carrying a mutant human superoxide dismutase 1 (SOD1) variant and regarded as an animal model for ALS as well as in post‐mortem tissue of ALS patients. NLRP3 and its molecular components as well as IL1β were already detectable in SOD1 mice at a pre‐symptomatic stage after 9 weeks and further increased in 14 week old animals. Spinal cord astrocytes were identified as the major cell type expressing NLRP3 components. In human ALS tissue, we also found increased NLRP3, ASC, IL18 and active caspase 1 levels compared to control patients. Our findings suggest that astroglial NLRP3 inflammasome complexes are critically involved in neuroinflammation in ALS. GLIA 2015;63:2260–2273     

Chronic transplantation of olfactory ensheathing cells promotes partial recovery after complete spinal cord transection in the rat

The goal of this study was to ascertain whether olfactory ensheathing cells (OECs) were able to promote axonal regeneration and functional recovery when transplanted 45 days after complete transection of the thoracic spinal cord in adult rats. OECs promoted partial restitution of supraspinal pathways evaluated by motor evoked potentials and modest recovery of hindlimb movements. In addition, OEC grafts reduced lumbar reflex hyperexcitability from the first month after transplantation. Histological results revealed that OECs facilitated corticospinal and raphespinal axons regrowth through the injury site and into the caudal spinal cord segments. Interestingly, raphespinal but not corticospinal fibers regenerated long distances through the gray matter and reached the lower lumbar segments (L5) of the spinal cord. However, delayed OEC grafts failed to reduce posttraumatic astrogliosis. In conclusion, the beneficial effects found in the present study further support the use of OECs for treating chronic spinal cord injuries.     

Resveratrol Improves Motoneuron Function and Extends Survival in SOD1G93A ALS Mice

Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease that causes progressive paralysis and death due to degeneration of motoneurons in spinal cord, brainstem and motor cortex. Nowadays, there is no effective therapy and patients die 2–5 years after diagnosis. Resveratrol (trans-3,4′,5-trihydroxystilbene) is a natural polyphenol found in grapes, with promising neuroprotective effects since it induces expression and activation of several neuroprotective pathways involving Sirtuin1 and AMPK. The objective of this work was to assess the effect of resveratrol administration on SOD1^G93A ALS mice. We determined the onset of symptoms by rotarod test and evaluated upper and lower motoneuron function using electrophysiological tests. We assessed the survival of the animals and determined the number of spinal motoneurons. Finally, we further investigated resveratrol mechanism of action by means of western blot and immunohistochemical analysis. Resveratrol treatment from 8 weeks of age significantly delayed disease onset and preserved lower and upper motoneuron function in female and male animals. Moreover, resveratrol significantly extended SOD1^G93A mice lifespan and promoted survival of spinal motoneurons. Delayed resveratrol administration from 12 weeks of age also improved spinal motoneuron function preservation and survival. Further experiments revealed that resveratrol protective effects were associated with increased expression and activation of Sirtuin 1 and AMPK in the ventral spinal cord. Both mediators promoted normalization of the autophagic flux and, more importantly, increased mitochondrial biogenesis in the SOD1^G93A spinal cord. Taken together, our findings suggest that resveratrol may represent a promising therapy for ALS.     

Astrogliosis in ALS: possible interpretations according to pathogenetic hypotheses.

Literature findings on astrogliosis of the spinal cord and of the cortex of sporadic (SALS) and familial (FALS) cases of amyotrophic lateral sclerosis (ALS) and of SOD1 transgenic mice are analysed and compared with those of 50 autopsied personal cases of ALS. In the spinal cord, astrogliosis is definitely evident in the anterior horns and much less in anterior and lateral funiculi and dorsal horns. In the cortex reactive astrocytes show a distribution similar to that of ageing brains and in ischaemia and cannot be directly put in relation with neuronal loss. In the spinal cord of transgenic mice the evidence suggests a primary astrocytic response. The findings in human ALS, especially those of the cortex, are consistent with this hypothesis.     

Extracellular mutant SOD1 induces microglial‐mediated motoneuron injury

Through undefined mechanisms, dominant mutations in (Cu/Zn) superoxide dismutase‐1 (mSOD1) cause the non‐cell‐autonomous death of motoneurons in inherited amyotrophic lateral sclerosis (ALS). Microgliosis at sites of motoneuron injury is a neuropathological hallmark of ALS. Extracellular mutant SOD1 (mSOD1) causes motoneuron injury and triggers microgliosis in spinal cord cultures, but it is unclear whether the injury results from extracellular mSOD1 directly interacting with motoneurons or is mediated through mSOD1‐activated microglia. To dissociate these potential mSOD1‐mediated neurotoxic mechanisms, the effects of extracellular human mSOD1^G93A or mSOD1^G85R were assayed using primary cultures of motoneurons and microglia. The data demonstrate that exogenous mSOD1^G93A did not cause detectable direct killing of motoneurons. In contrast, mSOD1^G93A or mSOD1^G85R did induce the morphological and functional activation of microglia, increasing their release of pro‐inflammatory cytokines and free radicals. Furthermore, only when microglia was co‐cultured with motoneurons did extracellular mSOD1^G93A injure motoneurons. The microglial activation mediated by mSOD1^G93A was attenuated using toll‐like receptors (TLR) 2, TLR4 and CD14 blocking antibodies, or when microglia lacked CD14 expression. These data suggest that extracellular mSOD1^G93A is not directly toxic to motoneurons but requires microglial activation for toxicity, utilizing CD14 and TLR pathways. This link between mSOD1 and innate immunity may offer novel therapeutic targets in ALS. © 2009 Wiley‐Liss, Inc.     

Radionuclide imaging of mesenchymal stem cells transplanted into spinal cord

In vivo tracking of stem cells implanted to spinal cord by radionuclide imaging was investigated. The high expression of transferrin receptor on human mesenchymal stem cells (hMSCs) was verified by flow cytometry, radioligand binding and immunofluorescence. Radiolabelled transferrin was chosen as a tracer for scintigraphic imaging of the hMSCs transplanted into spinal cord of rabbits. Comparative experiments with radiolabelled human serum albumin as tracer and PBS as graft as well as ex vivo autoradiography demonstrated the specific uptake of radiolabelled transferrin of hMSCs. hMSCs could be detected in vivo with radiolabelled transferrin targeting at cellular transferrin receptors at an early stage after transplantation into spinal cord.     

S100A6 overexpression within astrocytes associated with impaired axons from both ALS mouse model and human patients

Astrogliosis is one of the earliest pathological changes observed in neurodegenerative diseases in general and in amyotrophic lateral sclerosis (ALS) in particular. ALS is characterized by selective degeneration of motoneurons. There are 2 forms of the disease: sporadic ALS (SALS), comprising 90%-95% of cases, and familial ALS (FALS), comprising 5%-10% of cases. FALS is an age-dependent autosomal dominant disorder in which mutations in the homodimeric enzyme Cu/ Zn superoxide dismutase 1 (SOD1) is linked to the disease. The animal model for this disease is a transgenic mouse expressing the mutated human SOD1(G93A) gene. Here we show by immunohistochemistry and double immunofluorescence that astrocytes located near impaired axons of motoneurons that were selectively programmed to die overexpressed S100A6, a Ca2+/Zn2+ binding protein able to translocate into the nucleus. Transgenic mice overexpressing the mutated human SOD1 gene and patients suffering from SALS showed this selective astrocytic S100A6 expression. For instance, the pyramidal tract could be macroscopically detected on S100A6-labeled spinal cord and brainstem sections from SALS patients. Transgenic mice overexpressing the non-mutated SOD1 gene did not overexpress S100A6, although glial fibrillary associated protein astrogliosis was seen. Although these results do not give any clue about the beneficial or detrimental role played by S100A6, its induction may be assumed to appropriately serve some function(s).     

Relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial ALS

Transgenic mice that highly over-express a mutated human CuZn superoxide dismutase (SOD1) gene [gly⁹³→ala; TgN(SOD1-G93A)G1H line] found in some patients with familial ALS (FALS) have been shown to develop motor neuron disease that is characterized by motor neuron loss in the lumbar and cervical spinal regions and a progressive loss of motor activity. The mutant Cu,Zn SOD exhibits essentially normal SOD activity but also generates toxic oxygen radicals as a result of an enhancement of a normally minor peroxidase reaction. Consequently, lipid and protein oxidative damage to the spinal motor neurons occurs and is associated with disease onset and progression. In the present study, we investigated the time course of microglial (major histocompatibility-II antigen immunoreactivity) and astrocytic (glial fibrillary acidic protein immunoreactivity) activation in relation to the course of motor neuron disease in the TgN(SOD1-G93A)G1H FALS mice. Four ages were investigated: 30 days (pre-motor neuron pathology and clinical disease); 60 days (after initiation of pathology, but pre-disease); 100 days (approximately 50% loss of motor neurons and function); and 120 days (near complete hindlimb paralysis). Compared to non-transgenic littermates, the TgN(SOD1-G93A)G1H mice showed significantly increased numbers of activated astrocytes (P < 0.01) at 100 days of age in both the cervical and lumbar spinal cord regions. However, at 120 days of age, the activation lost statistical significance. In contrast, microglial activation was significantly increased several-fold at both 100 and 120 days. We hypothesize that astrocytic activation may exert a trophic influence on the motor neurons that is insufficiently maintained late in the course of the disease. On the other hand, the sustained, intense microglial activation may conceivably contribute to the oxidative stress and damage involved in the disease process. If true, then agents which inhibit microglia may help to limit disease progression. GLIA 23:249–256, 1998. © 1998 Wiley-Liss, Inc.     

Spinal cord microglial cells and DRG satellite cells rapidly respond to transection of the rat sciatic nerve

Transection of the rat sciatic nerve induces retrograde changes in the dorsal root ganglia (DRG) neurons and in the motoneurons in the ventral grey matter of the lumbar L4-L6 spinal cord segments. In the ipsilateral dorsal grey matter and in the ipsilateral nucleus gracilis, transganglionic changes occur in the terminal fields of the centrally projecting axons of injured DRG neurons. As revealed by immunocytochemistry, the neuronal reactions were associated with a rapid proliferation and activation of microglial cells in the lumbar spinal cord as well as in the nucleus gracilis. Reactive microglial cells were detected as early as 24 h after sciatic axotomy. The microglial reaction had a maximum around day 7 postlesion and disappeared around 6 weeks after axotomy. In addition to light microscopy, activated, perineuronal microglia were identified by immuno-electron microscopy in the ventral grey matter. In the DRG, satellite cells constitutively expressed major histocompatibility complex (MHC) class II antigens. Sciatic axotomy led to a proliferation of satellite cells and an increased expression of MHC class II molecules in particular. This satellite cell reaction started 24 h after axotomy and continued to increase gradually until about 6 weeks after the lesion. Resident macrophages, detected in the DRG interstitial tissue by their expression of monocyte/macrophage markers, also reacted to sciatic axotomy. Our data suggest that (1) sciatic axotomy leads to a rapid microglial reaction in both the ventral and dorsal grey matter of the lumbar spinal cord and in the ipsilateral nucleus gracilis; (2) the immunophenotype of activated microglia following sciatic axotomy is comparable with that observed after axotomy of cranial nerves, e.g. the facial nerve; (3) satellite cells in DRG constitutively express MHC class II molecules; and (4) sciatic axotomy leads to a rapid activation of satellite cells and interstitial macrophages in the axotomized DRG.     

Sigma-1R Agonist Improves Motor Function and Motoneuron Survival in ALS Mice

Amyotrophic lateral sclerosis is a neurodegenerative disorder characterized by progressive weakness, muscle atrophy, and paralysis due to the loss of upper and lower motoneurons (MNs). Sigma-1 receptor (sigma-1R) activation promotes neuroprotection after ischemic and traumatic injuries to the central nervous system. We recently reported that sigma-1R agonist (PRE-084) improves the survival of MNs after root avulsion injury in rats. Moreover, a mutation of the sigma-1R leading to frontotemporal lobar degeneration/amyotrophic lateral sclerosis (ALS) was recently described in human patients. In the present study, we analyzed the potential therapeutic effect of the sigma-1R agonist (PRE-084) in the SOD1^G93A mouse model of ALS. Mice were daily administered with PRE-084 (0.25?mg/kg) from 8 to 16?weeks of age. Functional outcome was assessed by electrophysiological tests and computerized analysis of locomotion. Histological, immunohistochemical analyses and Western blot of the spinal cord were performed. PRE-084 administration from 8?weeks of age improved the function of MNs, which was manifested by maintenance of the amplitude of muscle action potentials and locomotor behavior, and preserved neuromuscular connections and MNs in the spinal cord. Moreover, it extended survival in both female and male mice by more than 15?%. Delayed administration of PRE-084 from 12?weeks of age also significantly improved functional outcome and preservation of the MNs. There was an induction of protein kinase C-specific phosphorylation of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor in SOD1^G93A animals, and a reduction of the microglial reactivity compared with untreated mice. PRE-084 exerts a dual therapeutic contribution, modulating NMDA Ca²⁺ influx to protect MNs, and the microglial reactivity to ameliorate the MN environment. In conclusion, sigma-1R agonists, such as PRE-084, may be promising candidates for a therapeutical strategy of ALS.     

Expression of cholinergic phenotype by embryonic ventral horn neurons transplanted into the spinal cord in the rat

Solid grafts of E12 embryonic spinal ventral horn were transplanted into motoneuron-depleted adult lumbar spinal cord in the rat. A muscle was implanted parallel to the vertebral column with its nerve inserted into the lumbar cord at the site of transplantation so as to provide a target for innervation by the grafted neurons. Previous retrograde labelling studies have shown that modest numbers of grafted motoneuron-like cells participate in the muscle's reinnervation and these are often found outside the graft within the host spinal cord. However, Nissl stained sections show that larger numbers of neurons survive within tissue recognisable as being of graft origin. In this study we have examined the expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) by neurons within the graft. These enzymes are involved in cholinergic neurotransmission and are characteristic of motoneurons. Thirty-four to seventy days following transplantation the grafts contained numerous neurons with acetylcholinesterase (AChE) activity. Different patterns of AChE staining were observed which probably reflected the degree of differentiation and maturation within the graft. AChE positive neurons were found in isolation or in groups resembling developing motor pools. Most of the AChE-positive neurons appeared immature with scant cytoplasm. However, neurons could be found which appeared relatively mature with a regularly shaped nucleus, prominent nucleolus and Nissl bodies. The grafts contained few AChE-positive axons and no dense plexuses of varicose fibres around the neurons such as are found around motoneurons in the mature ventral horn. Comparisons between the size of AChE-positive neurons in the graft and the size of AChE-positive neurons in the developing ventral horn found that the size of grafted neurons to be intermediate between the sizes of spinal motoneurons at E19 and P0. Far fewer grafted neurons were found to be immunoreactive for choline acetyltransferase (ChAT) than histochemically reactive for AChE. This was consistent with our findings in the spinal cord during normal development where we found that fixation and staining procedures which labelled adult motoneurons failed to reliably demonstrate ChAT immunoreactivety in normal motoneurons prenatally, although AChE histochemical reactivity could be demonstrated as early as E16. We conclude that the grafts contain numbers of immature motoneurons which fail to proceed beyond a certain stage of development, perhaps because of a failure to form appropriate efferent and afferent connections.