In this blog, I avoid studies that are not done on humans, firstly because the further away from humans and primates, the less sincere the scientific studies are. Then it is well known that the pre-clinical studies published are all extremely positive in order to attract investors (if possible private) who will finance clinical studies.

2,4-dinitrophenol (DNP)

The study examined here explores whether small doses of 2,4-dinitrophenol, a chemical used in the manufacture of pesticides and slimming drugs because it decreases the metabolic action of mitochondria, could help protect motor neurons, preserve muscle function and slow the progression of amyotrophic lateral sclerosis (ALS) in a mouse model of the disease. The scientists' guiding idea therefore seems to be to slow down the disease, by slowing down the metabolism. This seems absolutely counter-intuitive but the scientists assure that they have had good results. The effective dose of 2,4-dinitrophenol was very low (0.5–1 mg/kg, human equivalent dose 2.5–5 mg/day), making it safer for potential use in humans. Dinitrophenol acts as a proton transporter in the mitochondrial membrane, inhibiting oxidative phosphorylation of ATP and making energy production less efficient. This is because some of the energy that is normally produced from cellular respiration is wasted as heat. This inefficiency is proportional to the dose of dinitrophenol that is absorbed. Thus, as the dose increases, energy production becomes less efficient: metabolism is then activated - more fat is burned - to compensate for the inefficiency and meet energy demands.

The researchers used hSOD1G93A mice, a common model of ALS, to test the effects of 2,4-dinitrophenol on motor skills, muscle strength, and disease progression. However, it is important to remember that most ALS patients do not have this mutation and that its notoriety is simply because it was the first mutation to be associated with ALS and that for more than 10 years (from 1993 to 2006) no other deleterious mutations were discovered in ALS.

Results

Mice treated with microdoses of 2,4-dinitrophenol (0.5–1 mg/kg) showed better motor coordination and better results on tests measuring muscle strength, compared to untreated mice. Early treatment initiation (before symptoms appeared) delayed the onset of motor decline, while late treatment initiation (after symptoms appeared) improved motor skills (which were already impaired by the disease) and slowed disease progression. Treated mice retained their ability to perform tasks such as running at 20 cm/s for longer than untreated diseased mice. In some cases, the mice regained lost motor skills, which is unusual in ALS research.

Although ALS damages the connections between nerves and muscles (neuromuscular junctions), treatment with 2,4-dinitrophenol preserved these connections. Treated mice retained a higher number of motor units (groups of muscle fibers controlled by a single neuron), indicating that motor neuron loss was reduced.

Reduced Cellular Stress

2,4-dinitrophenol reduced oxidative stress, a harmful process linked to the progression of ALS. This was demonstrated by lower levels of damaged proteins in treated muscles. While ​​slowing down an ALS patient’s metabolism seems pretty criminal, reducing cellular stress is a very good idea.

The drug also reduced inflammation and activated pathways (like Akt/mTOR) involved in muscle growth and repair.

How would 2,4-dinitrophenol work?

2,4-dinitrophenol acts on mitochondria, the energy producers of cells, by slightly lowering their membrane potential (∆Ψm). This mild “uncoupling” reduces the production of harmful reactive oxygen species (ROS), which can damage cells. 2,4-dinitrophenol may also help clear damaged mitochondria and promote the formation of healthy mitochondria, further protecting neurons and muscles.

Future Directions

While the mouse results are promising, translating these findings to humans requires more research. The study also highlighted some limitations:

• It focused on one type of ALS model (hSOD1G93A) that affects less than 2% of patients.

• It did not address potential differences in drug responses between male and female mice, which could be significant.

• Administering a metabolic reducer to ALS patients still seems like a dangerous notion.

In recent years, research has shown that physical exercise has many benefits for the entire body, beyond muscle growth. There has been much discussion in recent years about the value of physical activity programs in the case of Alzheimer's or Parkinson's disease.

Researchers at MIT wrote about experiments that might lend credence to the idea that mechanical stimulation could one day be beneficial in ALS or other MND diseases.

Muscles release various biochemical factors, called myokines, which circulate in the bloodstream. These molecules establish a kind of dialogue between different tissues and this allows them to collectively adapt to a new environment. Myokine receptors are found in muscle, fat, liver, pancreas, bone, heart, immune system and brain cells. The location of these receptors reflects the fact that myokines have multiple functions. First, they are involved in the metabolic changes associated with exercise, as well as in the metabolic changes that follow adaptation to training. To study these effects specifically on motor neurons, the cells that transmit movement commands from the brain to muscles, MIT researchers developed a series of in vitro systems where they could precisely control and observe the effects of muscle contractions.

In 2023, Raman and his colleagues reported that they could restore mobility in mice that had suffered traumatic muscle injury by first implanting muscle tissue at the site of the injury and then exercising the new tissue by repeatedly stimulating it with light. Over time, they found that the exercised graft helped the mice regain motor function, reaching levels of activity comparable to those of healthy mice. This meant not only that the new muscle tissue had become functional, but also that there were somehow new connections between the lower motor neurons and the new muscles. In other words, the exercise was not only beneficial for the new muscle but also for the local motor neuron that develops synapses to connect to muscle fibers.

Then the group wondered: Could exercise’s purely physical impacts have a similar benefit on motor neurons?

This claim was met with some skepticism.

So the MIT team designed experiments in which the neurons were repeatedly pulled back and forth, similar to the way muscles contract and expand during exercise. Angel Bu is the first author, while Ritu Raman is the senior author; the other authors are from MIT’s Department of Mechanical Engineering and MIT’s Koch Institute for Integrative Cancer Research. The authors matured a set of motor neurons on a gel that was a kind of carpet into which they embedded tiny magnets. They then used an external magnet to shake the carpet—and the neurons—back and forth. In this way, they made the neurons work, for 30 minutes a day.

The researchers demonstrated that the physical force generated during muscle contraction has direct mechanical effects on motor neurons, promoting growth in both a biochemical and mechanical way. And the two effects, myokine release and motor neuron work, have similar effects.

These results could have important implications for the treatment of motor neuron diseases, such as amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy, where motor neurons progressively lose their function. Therapies that mechanically stimulate muscle contractions could encourage nerve growth and regeneration, potentially slowing the progression of these diseases or facilitating recovery after nerve damage.

Of course, this study is extremely preliminary, it is carried out in vitro. We cannot even talk about micro-organs. This is basic research indeed, the authors didn't talk about applications in ALS/MND diseases. Another limitation of this study is that the authors did not explore a wide variety of mechanical or biochemical stimulation protocols with different frequencies, magnitudes, and durations.

If these mechanisms were confirmed in preclinical studies with primates and then clinical studies with humans, exercise protocols could be refined to maximize the biochemical and mechanical benefits for motor neurons, potentially improving motor function or slowing disease progression in patients with motor neuron diseases.

For example, patients could benefit from muscle stimulation devices that could help maintain or even regenerate motor neuron pathways. Mechanical muscle stimulation could indeed be a way to mimic the combined biochemical and mechanical effects of muscle contractions in patients with weakened muscles, potentially helping to slow neuronal degeneration or even stimulate nerve repair.

Electrical stimulators already exist for physiotherapy and rehabilitation, and future protocols could incorporate fine-tuning to produce exercise-like contractions that not only provide muscle benefits but also encourage motor neuron activity. Given that MND patients vary greatly in progression and severity, personalized electrical stimulation programs would be essential. Clinicians could develop individualized regimens, perhaps informed by biomarkers or real-time feedback, to maximize neuron growth-promoting effects without overloading the system.

Future research could test whether electrical muscle stimulation in MND patients produces benefits similar to biochemical and mechanical stimulation observed in vitro. If effective, this could lead to new physiotherapy protocols or devices aimed at improving the quality of life and motor function of people with MND.

Until Tofersen from Biogen, ALS drugs were only slightly slowing the disease. Tofersen aims to lower the levels of a specific mutation of SOD1. This therapy is probably a lifeboat for the patients with the matching SOD1 mutation. It's certainly slowing the disease progression, but only for a tiny portion of ALS patients less than 2%.

Not all patients with the right SOD1 mutation react to Tofersen similarly.

• People have two SOD1 genes in their cells and sometimes only one allele is mutated and the other can make up for the defective one. There are also other proteins named SOD2 and SOD3 that can assume partly the functions of SOD1.

• In the worst case with ASO medications, ALS patients are merely exchanging a defective protein with a lower production of the same protein. This is not a great perspective, a genetic therapy that makes cells produce the correct version of SOD1 would be better. Yet genetic therapies have their share of problems, including low efficiency and increased cancer risk.

All members of this protein family, transform a byproduct of the mitochondrial electron transport chain, into hydrogen peroxide and diatomic oxygen. Yet those two chemical species are themselves quite reactive. SOD1 is located in the cytoplasm of cells, SOD2 in their mitochondria, and SOD3 is extracellular.

In that case, where there is still one functional allele, it would be nice to check SOD1 levels in the body of patients treated with Tofersen. If it's too low (because of Tofersen) the therapy renders itself ineffective. This could be done by analyzing blood, but scientists reckon that checking SOD1 in CSF would be a better idea.

The discovery of children developing SOD1 Deficiency Syndrome (ISODDES) which is characterized by injury to the motor system, suggests that a too-low SOD1 antioxidant activity may be deleterious in humans. Measuring SOD1 activity in cerebrospinal fluid (CSF) in Tofersen-treated patients is recommended but difficult due to low concentration and the presence of the isoenzyme SOD3.

To assess SOD1 activity, the scientists propose to remove SOD3 from CSF samples with antibodies and subsequently measure the SOD1 activity. To propose this as a standard procedure for human beings is a bit weird. Repeatedly piercing the membranes that protect the spinal cord sounds like an unhealthy proposal, particularly if it's done to patients having motor neuron disease.