Jacalyn McHugh

GDNF secreting human neural progenitor cells protect dying motor neurons, but not their projection to muscle, in a rat model of familial ALS

Background Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by rapid loss of muscle control and eventual paralysis due to the death of large motor neurons in the brain and spinal cord. Growth factors such as glial cell line derived neurotrophic factor (GDNF) are known to protect motor neurons from damage in a range of models. However, penetrance through the blood brain barrier and delivery to the spinal cord remains a serious challenge. Although there may be a primary dysfunction in the motor neuron itself, there is also increasing evidence that excitotoxicity due to glial dysfunction plays a crucial role in disease progression. Clearly it would be of great interest if wild type glial cells could ameliorate motor neuron loss in these models, perhaps in combination with the release of growth factors such as GDNF. Methodology/Principal Findings Human neural progenitor cells can be expanded in culture for long periods and survive transplantation into the adult rodent central nervous system, in some cases making large numbers of GFAP positive astrocytes. They can also be genetically modified to release GDNF (hNPCGDNF) and thus act as long-term ‘mini pumps’ in specific regions of the rodent and primate brain. In the current study we genetically modified human neural stem cells to release GDNF and transplanted them into the spinal cord of rats over-expressing mutant SOD1 (SOD1G93A). Following unilateral transplantation into the spinal cord of SOD1G93A rats there was robust cellular migration into degenerating areas, efficient delivery of GDNF and remarkable preservation of motor neurons at early and end stages of the disease within chimeric regions. The progenitors retained immature markers, and those not secreting GDNF had no effect on motor neuron survival. Interestingly, this robust motor neuron survival was not accompanied by continued innervation of muscle end plates and thus resulted in no improvement in ipsilateral limb use. Conclusions/Significance The potential to maintain dying motor neurons by delivering GDNF using neural progenitor cells represents a novel and powerful treatment strategy for ALS. While this approach represents a unique way to prevent motor neuron loss, our data also suggest that additional strategies may also be required for maintenance of neuromuscular connections and full functional recovery. However, simply maintaining motor neurons in patients would be the first step of a therapeutic advance for this devastating and incurable disease, while future strategies focus on the maintenance of the neuromuscular junction.

Human neural progenitors deliver glial cell line-derived neurotrophic factor to parkinsonian rodents and aged primates

Glial cell line-derived neurotrophic factor (GDNF) has been shown to increase the survival and functioning of dopamine neurons in a variety of animal models and some recent human trials. However, delivery of any protein to the brain remains a challenge due to the blood/brain barrier. Here we show that human neural progenitor cells (hNPC) can be genetically modified to release glycosylated GDNF in vitro under an inducible promoter system. hNPC-GDNF were transplanted into the striatum of rats 10 days following a partial lesion of the dopamine system. At 2 weeks following transplantation, the cells had migrated within the striatum and were releasing physiologically relevant levels of GDNF. This was sufficient to increase host dopamine neuron survival and fiber outgrowth. At 5 weeks following grafting there was a strong trend towards functional improvement in transplanted animals and at 8 weeks the cells had migrated to fill most of the striatum and continued to release GDNF with transport to the substantia nigra. These cells could also survive and release GDNF 3 months following transplantation into the aged monkey brain. No tumors were found in any animal. hNPC can be genetically modified, and thereby represent a safe and powerful option for delivering growth factors to specific targets within the central nervous system for diseases such as Parkinsons.

Intermittent Hypoxia and Stem Cell Implants Preserve Breathing Capacity in a Rodent Model of Amyotrophic Lateral Sclerosis

RATIONALE Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. OBJECTIVES To preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end stage. METHODS SOD1(G93A) rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. MEASUREMENTS AND MAIN RESULTS The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. CONCLUSIONS AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.

Sexual dimorphism in disease onset and progression of a rat model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease causing the progressive loss of brain and spinal cord motor neurons. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Recently, transgenic rats overexpressing mutant forms of the human SOD1 (hSOD1) gene have been established as a valuable disease model of ALS. Here we show that sexual dimorphism in disease onset is also observed in hSOD1G93A transgenic rats. Disease onset was consistently earlier in male than in female hSOD1G93A rats. We also found that hSOD1G93A male rats lost weight more rapidly following disease onset compared to hSOD1G93A females. Furthermore, we tested locomotor function using the Basso‐Beattie‐Bresnahan (BBB) rating scale and a beam walking test. We found that motor dysfunction started earlier in males than in females but progressed similarly in the two sexes. These results have important implications for future experimentation and therapeutic development using the rat model of ALS.

Investigation into a role for the primitive streak in development of the murine allantois.

Despite its importance as the source of one of three major vascular systems in the mammalian conceptus, little is known about the murine allantois, which will become the umbilical cord of the chorio-allantoic placenta. During gastrulation, the allantois grows into the exocoelomic cavity as a mesodermal extension of the posterior primitive streak. On the basis of morphology, gene expression and/or function, three cell types have been identified in the allantois: an outer layer of mesothelial cells, whose distal portion will become transformed into chorio-adhesive cells, and endothelial cells within the core. Formation of endothelium and chorio-adhesive cells begins in the distal region of the allantois, farthest from the streak. Over time, endothelium spreads to the proximal allantoic region, whilst the distal outer layer of presumptive mesothelium gradually acquires vascular cell adhesion molecule (VCAM1) and mediates chorio-allantoic union. Intriguingly, the VCAM1 domain does not extend into the proximal allantoic region. How these three allantoic cell types are established is not known, although contact with the chorion has been discounted. In this study, we have investigated how the allantois differentiates, with the goal of discriminating between extrinsic mechanisms involving the primitive streak and an intrinsic role for the allantois itself. Exploiting previous observations that the streak contributes mesoderm to the allantois throughout the latters early development, microsurgery was used to remove allantoises at ten developmental stages. Subsequent whole embryo culture of operated conceptuses resulted in the formation of regenerated allantoises at all time points. Aside from being generally shorter than normal, none of the regenerates exhibited abnormal differentiation or inappropriate cell relationships. Rather, all of them resembled intact allantoises by morphological, molecular and functional criteria. Moreover, fate mapping adjacent yolk sac and amniotic mesoderm revealed that these tissues and their associated bone morphogenetic protein 4 (BMP4) did not contribute to restoration of allantoic outgrowth and differentiation during allantoic regeneration. Thus, on the basis of these observations, we conclude that specification of allantoic endothelium, mesothelium and chorio-adhesive cells does not occur by a streak-related mechanism during the time that proximal epiblast travels through it and is transformed into allantoic mesoderm. Rather, all three cell-types are established by mechanisms intrinsic to the allantois, and possibly include roles for cell age and cell position. However, although chorio-adhesive cells were not specified within the streak, we discovered that the streak nonetheless plays a role in establishing VCAM1s expression domain, which typically began and was thereafter maintained at a defined distance from the primitive streak. When allantoises were removed from contact with the streak, normally VCAM1-negative proximal allantoic regions acquired VCAM1. These results suggested that the streak suppresses formation of chorio-adhesive cells in allantoic mesoderm closest to it. Together with previous results, findings presented here suggest a model of differentiation of allantoic mesoderm that invokes intrinsic and extrinsic mechanisms, all of which appear to be activated once the allantoic bud has formed.

Multiple developmental roles of Ahnak are suggested by localization to sites of placentation and neural plate fusion in the mouse conceptus

Ahnak is a gigantic (700 kD) phosphoprotein with a unique structure whose expression and cellular localization are dynamically regulated during cell cycle progression. Here, we report that Ahnak is localized to sites of major morphogenesis during mouse placentation and neurulation. Ahnak was found in: (i) derivatives of trophectoderm, including chorionic ectoderm prior to and during union with the ectoplacental cone, presumptive syncytiotrophoblast cells in the chorionic labyrinth, and giant cells at the trophoblast-uterine interface; (ii) the allantois prior to, during, and after union with the chorion; and (iii) the tips of the neural plate during formation of the neural tube. On the basis of these observations, we suggest that Ahnak may play heretofore unrecognized roles in tissue union during normal mouse development.

Gonadectomy and dehydroepiandrosterone (DHEA) do not modulate disease progression in the G93A mutant SOD1 rat model of amyotrophic lateral sclerosis

Abstract Epidemiological studies have shown a higher incidence of amyotrophic lateral sclerosis (ALS) in men than women. Interestingly, there are clear gender differences in disease onset and progression in rodent models of familial ALS overexpressing mutated human superoxide dismutase-1 (SOD1-G93A). In the present study we sought to determine whether the alterations of serum steroid levels by gonadectomy or chronic treatment of neuroprotective neurosteroids can modulate disease onset and progression in a rat model of ALS (SOD1-G93A transgenic rats). Presymptomatic SOD1-G93A rats were gonadectomized or treated with a neurosteroid dehydroepiandrosterone (DHEA) using silastic tubing implants. Disease onset and progression of the animals were determined by the routine analyses of locomotor testing using the Basso-Beattie-Bresnahan (BBB) score. Although sexual dimorphism was observed in intact and gonadectomized SOD1-G93A rats, there was no significant effect of gonadectomy on disease onset and progression. DHEA treatment did not alter disease progression or survival in SOD1-G93A rats. Our results indicate that gonadal steroids or neurosteroids are not one of the possible modulators for the occurrence or disease progression in a rat model of ALS. Further analysis will be necessary to understand how sexual dimorphism is involved in ALS disease progression.

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.