ALS is a progressive, fatal motoneurone disease, ultimately leading to paralysis and respiratory failure within 3-5 years. There is currently only one FDA-approved drug, Riluzole, but its lack of disease prolongation combined with the aggressive disease nature means identifying new treatments is essential. This review highlights 3 of the most current and promising research areas.
Recent Phase 1 Clinical trials have proven safety of stem cell (SC) implantation in humans. Parallel rodent SC models show positive results in both decelerating disease progression and promoting anti-inflammatory neuronal protection. Supplementary use of growth factors also shows potential regarding motoneurone survival and dendrite length in cultures, and survival rates in mouse models.
Knockout of glial xC- glutamate anti-porter significantly reduces excessive glutamate levels in neurones by 70%, compared to xC- / microglia. Knockout also reduces levels of pro-inflammatory markers. These findings highlight vital role of xC- system in reducing neuronal glutamate excitotoxicity.
Antisense technologies effectively reduced SOD1 protein and mRNA levels, consistent in CSF and brain of SOD1 rodent cortices. This supports SOD1 as a good biomarker for future antisense studies.
Overall, there is promising research being conducted. However improvements in clinical trial techniques must be addressed in order to reliably compare findings from future studies, and allow identification of a cure in the future.
Summary word count: 213
Also known as ‘Lou Gehrig’s Disease’, Amyotrophic Lateral Sclerosis (ALS) involves loss of upper and lower motoneurones from the brainstem and spinal cord. Symptoms progress from difficulty in limb movement to paralysis, and finally respiratory failure, the biggest cause of death in ALS. With a prevalence of approx. 2:100,000 and average onset age of 55 years, death usually occurs 3-5 years after onset.
Although first identified in 1869 by Jean-Martin Charcot, there remain no conclusive disease causes. The disease is classified into 2 types: Familial ALS (fALS), the inherited form, is responsible for approx. 10% of all cases. There are a handful of genetic mutations linked to fALS, including: C90RF72, TDP-43, FUS, Ubiquilin-2, and currently most relevant in disease-models, Cu2 /Zn2 Superoxide Dismutase (SOD1). Sporadic (sALS) form comprises the majority of cases, and this unknown nature of the disease makes targeted treatments challenging.
Riluzole is currently the only FDA approved treatment for ALS, increasing life expectancy by 2 months. In 2011, ‘Nuedexta’ was also approved as a treatment for pseudobulbar effects in ND diseases. Patients must otherwise rely on palliative care to improve quality of life. This review will focus on the most current and widely researched areas. Proposed mechanisms of disease are beyond the scope of this review, but can be found in a review by Cleveland and Rothstein (2001).
Stem Cell Therapy
Due to its infamous potential, stem cell (SC) therapy is perhaps the most widely researched treatment area. SC therapy aims to improve symptoms rather than cure the disease, by either targeting re-growth of neurones or promoting their survival. Here we will focus on clinical and pre-clinical SC trials in the last 5 years.
Direct neuronal replacement
One proposed treatment method is to replace dying motoneurones with SCs. Recent Phase 1 Clinical Trials by Glass et al., (2012) and Feldman et al., (2014) studied the safety of lumbar and cervical SC injections, respectively. Both trials found good patient tolerance and sufficient safety to continue with future trials.
Feldman et al. also used histochemical analysis to address concerns over unpredictable SC migration, by highlighting successfully transplanted SCs in spinal cord slices, Figure 1.
Figure 1. B) Cross-section of spinal cord highlighting non-native cells C) Close-up shows morphology of cells consistent with pre-implanted SCs, indicative of successful transplantation into spinal cord. [Adapted from Feldman et al., 2014]
Despite multiple limitations to the experimental technique, such as absence of a control group, results indicated early SC transplantation has a good chance of slowing disease progression in ALS patients, as 50% of patients showed improvement in 6-15 month post-trial check-ups. The corresponding Phase II Trial commenced in September 2013, and is due for completion this month.
Figure 2 shows a previous study by Karussis et al., (2010) where SC injection leads to a significant increase in immune-regulatory cells (CD4 /CD25 ) and an overall decreased immune response.
Figure 2. Levels of neuronal cell inflammatory markers following injection of SCs
[Adapted from Karussis et al., 2014]
Reduction in immune response over 24hrs was in fact greater than seen in immunomodulatory medicines, suggesting additional mode of action for SC therapy.
Neuronal survival via growth factor delivery
Unsuccessful trials in the late 1990s to treat ALS with growth factors (GFs) prompted further studies into appropriate CNS targeting. Development of the SOD1-mutant rat model allowed Suzuki et al., in 2008, to address these delivery issues in a study using SCs as GF vectors. They found GF delivery to mid-stage SOD1 rodents showed increased neuromuscular connections, and a lifespan increase of 28 days, possibly due to reduced neuronal loss.
Viral vectors for trophic factors (TFs) provide an alternative delivery route, and in 2010, Dodge et al., carried out mouse embryonic-SC motoneurone studies in which expression of TFs IGF-1 and VEGF-165 using viral vector, AAV4, allowed successful delivery of TFs to entirety of CNS. This slowed MN decline and increased mouse survival. Figure 3 shows initial culture studies using mouse-derived embryonic motoneurone SCs, showing clear protective action on neurones.
Figure 3. A) 70% of motoneurones died in control, GFP-CM, compared to high survival with IGF-1-CM/VEGF-CM. B,C) IGF-1-CM/VEGF-CM treated motoneurones showed increased neurite length and survival rates compared to control. [Image from Dodge et al., 2010]
Subsequent mouse studies showed increased survival and decelerated reduction in hindlimb grip-strength and stamina on the rotarod, seen in Figure 4.
Figure 4. A,C,E) Mouse studies depicting neuroprotective action of TFs, IGF/VEGF vs control. [Image adapted from Dodge et al., 2010]
It is worth noting that combined delivery of both TFs showed no synergistic effect, probably due to their affecting the same pathway. Subsequently, in 2013, Krakora et al., modified human mesenchymal SCs to further investigate synergistic effect of combined GFs. A synergistic effect between GDNF VEGF was seen due to their action on different signalling cascades. This shows promise for future studies into improved neuronal survival.
Phase-1 clinical-trials have shown safety of SC injection into CNS with promising, if unreliable, patient outcomes. The mechanism of improvement still unidentified, but hints at inflammatory regulation in neural protection may open an interesting avenue. GF application shows further potential based on rodent/mice studies with a proven effect at slowing disease progression and neuronal loss. SCs make suitable GF vectors as can be made to express/over-express GFs. Combining GF models with SC vectors for targeted delivery requires further exploration. Future trials must consider frequency, dose and administration technique.
Mouse ALS models by Beers et al., (2011) and Liao et al., (2012) indicate microglia conversion from M2 (anti-inflammatory) to M1 (pro-inflammatory) state during disease. Believed to be due to glutamate toxicity, therapeutic work should focus on reducing excessive neuronal glutamate level, and reducing resultant pro-inflammatory response.
In 2014, Mesci et al., studied the xC- system; a glial antiporter exchanging cysteine for glutamate release, causing increased neuronal glutamate. The study aimed to show blocking xC- would reduce excessive glutamate release and affect M1/M2 state, to reduce inflammation.
xCT (transporter gene) -/- mouse microglial studies demonstrated a significant 70% reduction in glutamate release compared to XC- / . Furthermore, Figure 5 shows significantly increased pro-inflammatory factors in xC- / mice microglia compared to -/-, hinting at a shift towards the M1 microglial phenotype via xC-.
Figure 5. A-E) Levels of M1 pro-inflammatory factors in xCT -/- vs / mouse microglia
[Image from Mesci et al., 2014]
Interestingly, Mesci et al., also noted a 10-fold increase in anti-inflammatory M2 marker levels at pre-symptomatic phase in -/- mice which drops off at disease onset, indicating M1/M2 shift upon disease onset. Encouragingly, -/- microglia showed significant increase in motoneurone survival in -/- vs / microglia, at 45% and 35% survival, respectively. This is indicative of a less neurotoxic environment.
Finally, xC- -/- SOD1 mutated mice showed an overall deceleration in disease progression, shown by increased survival rates following advanced disease stage (20% weight loss) in Figure 6.
Figure 6. Survival in advanced ALS in xC- / and -/- SOD1 mice
These anti-excitotoxicity findings are consistent with the action of Riluzole. Future drugs may target xC- system, however current antagonists are poorly specific and available to brain. Identification of a more suitable antagonist would be a good priority before further clinical trials.
Antisense oligonucleotides (ASOs) bind to specific mRNA sequences to cause mRNA degradation. In 2013, Leah et al., conducted studies in SOD1 rodents and human subjects with neurodegenerative diseases. SOD1-targeting ASOs were introduced to subjects to reduce SOD1 levels.
They found both SOD1 mRNA and protein fell by 69±4% and 48±14%, respectively, in rodent cortices. Interestingly, this matched reduced protein levels by 42±14% in rodent CSF, indicating CSF levels are a good measure of levels in brain.
Unfortuntely, SOD1 cannot be a specific ALS marker due to its presence in other neurodegenerative diseases. However, its observed constant levels over time supports SOD1 as a good biomarker in indicating efficacy of antisense technologies and its effective targeting by ASOs may be useful in measuring brain SOD1 levels via CSF levels in future studies.
A Phase 1 Clinical Trial by Miller et al., (2013) to determine safety of single-dose intrathecal injection of ASOs found no safety concerns. However, liver cancer and neuropathy was previously seen in cases of chronic low SOD1 levels (Elchuri et al., 2005), so long-term dosage effects must be carefully monitored.
This review was restricted to three main research areas, but areas such as susceptibility gene identification, oxidative stress and protein misfolding, are also being explored.
Current research shows promise, especially in SOD1 rodent models and positive safety data from Phase 1 Trials. SCs continue to have vast potential, and when combined with GFs have shown encouraging effects on disease progression in rodent models.
Reliability of these studies, however, must be improved in order to draw accurate conclusions and compare findings from related studies. Ideally, a ‘standard trial protocol’ should be implemented. Trials must also consider long-term effects of reduced SOD1 levels (in antisense techniques) and immunosuppressant use (with stem cells). Some issues may possibly be alleviated by recent development of new autologous SC models (Meyers et al., 2014).
In short, current ALS treatments remain palliative care and Riluzole, but with new developments continuously emerging there is definitely an exciting research landscape ahead. In October 2014, ALSA requested Phase II Trial proposals to accelerate work in this area, meaning that the race is on to find suitable ALS treatment that may help patient prognosis in the future.
Word count: 1573
Effects of Allelopathy in Plants
What do we learn about the effect of allelopathy in plants, in particular the effects that garlic and broccoli have on the growth of other plants?
1. State the problem. We are going to find out whether garlic volatiles or broccoli volatiles affect celery growth and if they do, how much do they hinder their growth.
2. State your hypothesis. The presence of garlic volatiles inhibits celery growth, but the presence of broccoli volatiles has no affect.
3. What are you going to measure (dependent variable)? Growth of celery seeds in the presence of garlic volatiles. Growth of celery seeds in the presence of broccoli volatiles.
4. What experimental variable are you investigating? Growth of celery seeds without garlic present. Growth of celery seeds without broccoli present.
5. What treatment(s) will you use? Group PG1: Celery with garlic, Group PG2: Celery without garlic Group PB1: Celery with broccoli, Group PB2: Celery without broccoli
6. Do you need a control? If not, then why not? If so, what is it? We do need a control group. If we do not have a control group, we will not be able to measure the growth of “celery with garlic” (and “celery with broccoli”) and compare it to anything else. The control group for each study allows us to make a good comparison.
7. What nuisance variable(s) will you be stabilizing? How will you stabilize each one? Temperature, water, sunlight. Temperature of all seeds will be the same because they are place in a common location. Sunlight will be present for all seeds and absent for all seeds at the same time. Water: when added, will be added in the same amount at the same time for all seeds.
8. What sample size will you use? A sample size of 20 seeds of celery will be used in each of the four groups.
9. Will you replicate your study? If yes, then how? If not, then why not? We will replicate our study because each group consists of a sample size of 20 seeds, so we are replicating by comparing the results for 20 occurrences.
We will measure the growth of celery in the presence and absence of garlic volatiles. We expect that growth will be inhibited by garlic volatiles. We will also measure the growth of celery in the presence and absence of broccoli volatiles, but we expect broccoli to have no effect.
Allelopathy (from the Greek, allelon, meaning another and pathos, which means to suffer) is a way some plants deal with competition.
Allelopathic plants release chemical substances which literally make other plants suffer. Some of these allelopathic plants store toxins in their leaves. When the leaves fall to the ground, the toxins are released. These toxins leach through the soil and are taken up by other nearby plants.
Alternately, allelopathic plants can release chemicals through their roots. These toxins travel through the soil where they can be absorbed by the roots of other plants.
Some allelopathic plants use gas warfare by releasing allelochemicals through small pores in their leaves and gassing nearby species.
We will conduct two experiments simultaneously. In one experiment, we will be using garlic volatiles to find out if the gases they emit negatively affect the growth of celery seeds. In the second experiment, we will use broccoli volatiles instead of garlic volatiles.
We took 4 petridishes, put a filter paper at the bottom of each and added about 2 ml of water to each one. In one petridish we placed a portion of crushed garlic in the center, placed in a piece of aluminum foil. This would be compared with a petridish in which the piece of aluminum foil had no crushed garlic on it. Similarly, we had a petridish with and without crushed broccoli. The labeling and contents of each pertridish were as follows:
PG1 = petridish with crushed garlic (experimental group 1) PG2 = petridish without crushed garlic (control group 1) PB1 = petridish with crushed broccoli (experimental group 2) PB2 = petridish without crushed broccoli (control group 2)
We carefully placed 20 celery seeds in each petridish, evenly distributed along the filter paper around the center. We covered all four petridishes and taped them along the sides, to minimize loss of moisture.
Over the next 2 weeks, we measured the growth of celery seeds in each petridish. If water had dried up, we added the same amount of water to each and continued to record the growth.
DATA AND RESULTS:
Scientific Inquiry Experiment Data Sheet: Page 1 of 2
EXPERIMENTAL GROUP 1 (PG1) CELERY SEEDS WITH GARLIC
Date of Observation Parameter measured Number of seeds germinated and growing Comments 01/07/10 no growth observed Less than 24 hours has passed 01/11/10 5 out of 20 have shoots 01/12/10 5 out of 20, a couple sprouts Added 1 ml water 01/13/10 8 out of 20, 5 have leaves 01/14/10 8 out of 20, 5 growing longer Added 1 ml water 01/19/10 6 out of 14, remaining seem rotten
CONTROL GROUP 1 (PG2) CELERY SEEDS WITHOUT GARLIC
Date of Observation Parameter measured Number of seeds germinated and growing Comments 01/07/10 no growth observed Less than 24 hours has passed 01/11/10 10 out of 20 have shoots 01/12/10 13 out of 20, all showed stems, separated from seeds Added 1 ml water 01/13/10 13 out of 20, stems growing longer 01/14/10 18 out of 20, stems intertwined Added 1 ml water 01/19/10 20 out of 20 all growing
Scientific Inquiry Experiment Data Sheet: Page 2 of 2
EXPERIMENTAL GROUP 2 (PB1) CELERY SEEDS WITH BROCCOLI
Date of Observation Parameter measured Number of seeds germinated and growing Comments 01/07/10 no growth observed Less than 24 hours has passed 01/11/10 13 out of 20 have little stems 01/12/10 19 out of 20, longer stems, more sprouts Added 1 ml water 01/13/10 16 out of 20, 11 sprouted and growing, 5 germinated w/no stems 01/14/10 16 out of 20 Added 1 ml water 01/19/10 17 out of 20
CONTROL GROUP 2 (PB2) CELERY SEEDS WITHOUT GARLIC
Date of Observation Parameter measured Number of seeds germinated and growing Comments 01/07/10 no growth observed Less than 24 hours has passed 01/11/10 17 out of 20 have long white stems with green leaves Very quick growth!!! 01/12/10 17 out of 20, all growing longer fast Added 1 ml water 01/13/10 17 out of 20, no room to grow, tangled 01/14/10 17 out of 20 Added 1 ml water 01/19/10 17 out of 20
It is very obvious that garlic volatiles adversely affected ability of celery seeds to grow. Even those that sprouted seemed to rot quickly.
As far as broccoli, the results are not so clear. Even though the seeds on the dish with broccoli grew slower than the one without broccoli, they did eventually grow and some grew better in broccoli than without broccoli. This part of the experiment would need to be repeated to come to a decision on whether broccoli inhibited the growth of celery seeds or not. It is even possible that broccoli may help celery to thrive.
Witt, Jon. Soc.. 1st ed. New York, NY: McGraw-Hill, 2008. 67-68. Print. (use this for format only)
http://www.suite101.com/blog/bobcajun/allelopathy Jan 19, 2008 Alleleopathy posted by Robert Dailey