25-Hydroxycholesterol May Accelerate Progression of ALS

Increased levels of 25-hydroxycholesterol is suspected to raise the number of neuron deaths and to accelerate the disease progression of ALS. This is according to a study that could possibly lead to new ALS therapies that are targeted by this molecule.

ALS or Amyotrophic Lateral Sclerosis is progressively degenerative neurological disease that leads to the death of the nerve cells responsible for voluntary muscle movement. The disease’s exact cause remains unknown and so there is no cure. Most diagnosed patients die within four years from the onset of the disease.

During the development and maintenance of the nervous system, several molecules contribute towards the pathology of Amyotrophic Lateral Sclerosis (ALS). Research has identified malfunctions of motor neuron function in ALS with the major culprits being protein misfolding, abnormal calcium metabolism, activation of proteases and nucleases, impaired energy production and excessive excitatory tone. Other factors have been suspected to instigate the pathogenesis: toxins, viral and nonviral infections, and autoimmune reactions.

Past studies have suggested the involvement of cholesterol and its intermediate molecules in the development and progression of the disease. These intermediate molecules are also known as hydroxycholesterol.

Cholesterol which is present in all cells of the body including those found in the central nervous system is synthesised de novo. During its biosynthesis, it is metabolised into various hydroxycholesterols (OHCs) which ensure that cholesterol homeostasis, signalling and cellular apoptosis are properly regulated.

The Cholesterol Biosynthesis Pathway

Cholesterol is fat-like substance, which is found in all cells of the body. The body needs it to make vital substances such as hormones and vitamin D, and other substances that are necessary for digestion. The body synthesises all the cholesterol it needs but also gets some from the foods we eat. The cholesterol travels throughout the body in lipoproteins that are made lipids on the inside and proteins on the outside.

Many people consider cholesterol to be generally a bad thing; that is not true. There are two kinds of lipoproteins that carry cholesterol: low-density lipoproteins (LDL) and high-density lipoproteins (HDL) and they blood levels determine the person’s health. High levels of LDL also referred to as “bad” cholesterol are known to clog arteries. HDL cholesterol is considered “good” because it carries cholesterol from other parts back to the liver for secretion.

The synthesis of cholesterol is an elaborate process. It starts with the synthesis of hydroxymethylglutaryl-CoA (HMG-CoA) using acetyl-CoA. The reactions occur in the cytosol of the cell. Acetyl-A is a product of the metabolism of either protein, fat or carbohydrate as a source of fat.

In the endoplasmic reticulum, the enzyme HMG-CoA reductase reduces HMG-CoA to mevalonate. Melvalonate is then further converted into different isoprene intermediates; that is, first into squalene and lanosterol which is finally converted into cholesterol.

Cholesterol is a type of lipid molecule that is biosynthesised by animal cells and it ensures the maintenance of the structural integrity and fluidity of the cell’s membrane. The liver is the main site for biosynthesis with 20% of the total daily cholesterol production; other sites include the adrenal glands, reproductive organs and the intestines.

The process of cholesterol biosynthesis is mostly regulated by the present cholesterol levels. When one consumes food loaded with cholesterol, there is a noticed decrease in the production process that originates from the cells, while lower intake levels have the opposite effect.

The study published in the Journal the Oncotarget, aimed at analysing the levels of the 24-, 25-, and 27- hydroxycholesterols in the serum and cerebrospinal fluids of ALS patients. It also sought to demonstrate the hydroxycholesterols’ role in the disease progression of ALS patients.

The Study

Thirty nine ALS patients aged between 20 and 80 years were recruited to participate in the study. The recruitment exercise ran between December 2009 and May 2012 excluding those on respiratory or feeding support. The participants were divided as follows: 30 were left untreated and nine were administered with the ALS drug Riluzole. Their results were compared with those of a group of 33 healthy controls.

Findings

1. Increased levels of hydroxycholesterols in ALS patients

The demographics, level of serum cholesterols, or body mass index of the participants and controls did not differ significantly. However the study found that the levels of 24- and 25-OHCs were higher in the group of patients who did not receive the drug treatment. The study findings also showed significantly higher levels of27- and 25-OHCs in the placebo group than in the control group.

The severity of the disease was measured by the revised ALS Functionality Rating Scale (ALSFRSr). It was found that the rate of disease progression was significantly correlated with the levels of 27-OHCs in the cerebrospinal fluid and 25- OHCs in the serum. Further analysis showed that only 25-OHC in the serum was significantly associated with the rate of disease progression.

2. Riluzole diminished the rate of neuron death induced by 25-OHC

Of the three studied hydroxycholesterols, 25-OHC induced the most severe neuronal apoptosis. 25-OHC also activated the GSK pathway which further induced cell apoptosis. However, when Riluzole was introduced, it attenuated the 25-OHC-induced apoptosis, activation of the GSK-3 pathway and neuronal death.

3. 25-OHC induced death of motor neurons via signalling of liver x receptor (LXR)

To aid in the evaluation of the mediative role of25-OHC in motor neuronal death via LXR signalling, a LXR antagonist 22(s)-OHC was introduced in ALS laboratory models alongside 25-OHC. The inclusion of the antagonist significantly reduced the 25-OHC induced motor neurons in direct proportion to the dose.

4. Upregulation of 25-Hydroxycholesterol Synthesis in ALS Mice

The cholesterol 25-hydroxylase (CH25H) and to a lesser extent cytochrome P450 3A4 (CYP3A4), found in the central nervous system, are responsible for generating 25-OHC. Likewise, 25-hydroxycholesterol 7-alpha-hydroxylase (CYP7B1) metabolises 25-OHC. In attempt to extrapolate a potential mechanism for the increased occurrence of 25-OHC in ALS patients, the researchers studied the mRNA expression of CH25H, CYP3A4, and CYP7B1 in the CNS of ALS gene expressing mouse models.

Before disease onset in the mice, the enzyme expressions in both the ALS and control mice were not different. However, when the symptoms set in, the expressions of the enzymes CH25H and CYP3A4 increased significantly. These changes were primarily observed in the brain tissue. The metabolizing enzyme CYP7B1 moderately increased in the brain and spinal cord of the symptomatic mice. Interestingly, these enzyme expressions were not observed in late symptomatic stages but only in the early stages.

Discussion

The study proved that 25-hydroxycholesterol is associated with the disease progression in ALS patients. The levels of 25-OHC in the cerebrospinal fluid and serum were higher in the patients without treatment than in those treated or the control group. A significant link was established between the levels of 25-OHC in the serum of ALS patients and the severity and progression rate of their disease. 25-OHC also triggered cellular death and activated the GSK-3 pathways in the ALS laboratory model. Finally, the 25-OHC synthesis and metabolism enzymes expression was significantly increased in the mice models; particularly in the early symptomatic stage. The findings of the study were in tandem with the case studies from existing literature.

Recently, a study found that 25-hydroxycholesterols in serum is higher in ALS patients than controls. According to the researchers at the Seoul National University Hospital, the difference did not reach statistical significance when compared to their study findings. They also acknowledged that the disparity in findings could be as a result of stringent patient inclusion criteria in their study. “Moreover, we have controlled the compounding effects of riluzole treatment on OHC level, measured the severity of patients by ALSFRSr, and analyzed the OHCCSF, as well as the OHC, which could have contributed the more robust and readily observable results in our study,” the team said.

Recently, a study found that 25-hydroxycholesterols in serum is higher in ALS patients than controls. According to the researchers at the Seoul National University Hospital, the difference did not reach statistical significance when compared to their study findings. They also acknowledged that the disparity in findings could be as a result of stringent patient inclusion criteria in their study. “Moreover, we have controlled the compounding effects of riluzole treatment on OHC level, measured the severity of patients by ALSFRSr, and analyzed the OHCCSF, as well as the OHC, which could have contributed the more robust and readily observable results in our study,” the team said.

Limitations of the Study

1. The number of patients was relatively small
2. 25-hydroxycholesterol cannot be the only factor responsible for the severity and progression of ALS. Even the treated group with relatively low levels eventually experienced disease progression
3. 2The researchers used serum and cerebrospinal fluid from the patients as controls as opposed to that from healthy individuals. This may not be a significant issue as the team excluded the inflammatory or degenerative diseases of the central nervous system from the controls
4. The OHCs levels in the serum and cerebrospinal fluid of the ALS animal models were not analysed because the concentrations were expected to be negligibly low

About the Medication Used in the Study

Riluzole is a drug used to slow the progress of ALS; most ALS patients eventually die of respiratory problems. ALS weakens many of the muscles of the body including those involved in breathing. The drug however, only delays the need for mechanical ventilation but does not cure the disease.
It is important to note the following before taking the medication:

• The drug is usually administered orally after every 12 hours; at the same time each day
• The doctor’s prescription and instructions should be followed carefully and clarification sought from the doctor or pharmacist where not understood
• The dosage should not be exceeded or taken less of against doctor’s directions
• Even after symptoms start easing up and you start feeling better, continue to take the drug. Do not stop before talking to your doctor

Precautions

1. Consult with your doctor or pharmacist in case of allergies to medication
2. Inform the doctor or pharmacist of all medication you are taking
3. Inform your doctor of any blood disorder, anaemia, liver or kidney disease
4. Women of childbearing age should notify their doctors if they are pregnant, plan to or are breast-feeding. If you get pregnant while on the medication, notify your doctor immediately
5. In case of any form of surgery while taking Riluzole, tell the doctor
6. Do not drive of operate machinery as the drug may cause drowsiness
7. Avoid alcohol
8. If you use tobacco products tell your doctor

Possible Side Effects of Riluzole

The use of Riluzole may trigger some side effects such as: tiredness, stomach pain, muscle weakness or aches, headache, dizziness, upset stomach, diarrhoea, or loss of appetite. If these symptoms persist or get worse, go see a doctor. However in case of severe effects like: depression, difficulty in breathing and fever, call your doctor immediately.

According to the researchers, their data showed the possibility of 25-OHC as an important mediator of ALS disease progression and severity by involving the activation of GSK-3 and neuronal apoptosis. These events could trigger the onset or accelerate the progression of the symptomatic stages of ALS. “Our results may reveal a novel ALS treatment target and provide insight into the involvement in of CNS cholesterol metabolism in ALS pathogenesis,” they concluded.

Conclusion

Hydroxycholesterols are involved in many functions of the body including, cytotoxicity, cellular cholesterol synthesis, and cell signalling. 24-OHC is associated with Alzheimer’s disease and its severity. 25-OHC has garnered the attention for its anti-inflammatory and pro-inflammatory mediator properties. “In addition to these important roles of OHCs, we speculated that 25-OHC might play a pathogenic role in ALS by mediating GSK-3ß activation, LXR signalling, and neuronal apoptosis,” said the author.

The severity of the disease in ALS patients was closely associated with the levels of 25-OHC in the serum and not in the cerebrospinal fluid. This correlation can be explained by the expression of the synthesising enzymes only being significant in the early symptomatic stages of the disease. The scientists suggest that temporary high expression could have interfered with the production of 25-OHC in the cerebrospinal fluid of late-stage patients.

The decrease of 25-OHC in the cerebrospinal fluid of the treated patients could be attributed to the anti-glutamatergic effects of the drug Riluzole. A previous study shed light on the increase of cholesterol and cholesterol oxidation products in the brain caused by glutamate excitotoxicity.

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Source: ALS News Today