Home-made Liposomal EGCG — a cost effective therapy for Autism, Parkinson’s, and Alzheimer’s? Plus alternative antioxidants — Whey protein and Liposomal vitamin C

A $30 ultrasonic jewellery cleaner can be repurposed to make inexpensive liposomal supplements

 

Today’s post is really one for those who prefer not to use prescription drugs to treat autism, or those that are just unable to access them. It is also one our longtime reader Ling might regard as MacGyver-esque (from the TV series following the adventures of Angus MacGyver, a secret agent armed with remarkable scientific resourcefulness to solve any problem out in the field using any materials at hand).

It is about increasing the bioavailability of OTC supplements (EGCG in today’s case, but applicable to many others) to get closer to achieving their often elusive health benefits in autism.

There are some effective OTC autism therapies, but most are not. This is why repurposing existing prescription drugs is likely necessary.

 

Liposomal

One of the big things in the supplement world at the moment is to call products “liposomal” and triple the price. The theory is that a preparation contains the active drug/supplement inside very tiny, fat-like particles. This form is easier for the body to absorb and allows more drug/supplement to get to the target area of the body, such as the brain. Liposomal drugs may have fewer side effects and should, in theory, work better than other forms of the drug.

This fatty encapsulation helps protect the active compound from degradation in the digestive system and improves its absorption through the gut. It can also enhance delivery to target tissues (like the brain) because liposomes can sometimes cross biological barriers more easily.

This should mean higher effectiveness with lower doses and potentially fewer side effects compared to non-encapsulated forms.

 

If you are interested in the details:

https://en.wikipedia.org/wiki/Liposome

“A liposome is a small artificial vesicle, spherical in shape, having at least one lipid bilayer. Due to their hydrophobicity and/or hydrophilicity, biocompatibility, particle size and many other properties, liposomes can be used as drug delivery vehicles for administration of pharmaceutical drugs and nutrients, such as lipid nanoparticles in mRNA vaccines, and DNA vaccines. Liposomes can be prepared by disrupting biological membranes (such as by sonication).

Liposomes are most often composed of phospholipids, especially phosphatidylcholine, and cholesterol, but may also include other lipids, such as those found in egg and phosphatidylethanolamine, as long as they are compatible with lipid bilayer structure. A liposome design may employ surface ligands for attaching to desired cells or tissues.”

 

 

  

By making your own liposomal supplements you will save a lot of money, compared to commercial ones and have access to an undegraded product. If you customize the recipe/ingredients thoughtfully, and carefully control the processing, the result might replicate some of the benefits seen in university studies. You might wonder why compounding pharmacies are not already doing this - maybe some are.

You can pretty much buy everything you need on Amazon. Once you have figured out your ingredients and decided how big a batch to make, it is no more complex than baking a cake.

 

Liposomal vitamin C and whey protein as therapies for oxidative stress

Oxidative stress is a core feature of most autism, particularly in the early years, and a feature of aging for everyone. Vitamin C is a natural antioxidant, but it is a water soluble vitamin that your body automatically regulates and excretes via urine. If you take mega-doses of a standard supplement it just goes down the toilet, it does not reach the bloodstream.

Intravenous vitamin C causes a large increase in levels in the blood. This can be used to treat sepsis and even mast cell activation syndrome (MCAS). It has potential in oncology (cancer treatment) because at high concentrations, vitamin C can act as a pro-oxidant, generating hydrogen peroxide that is selectively toxic to tumor cells.  

It has also been used for Ehlers-Danlos syndrome, fibromyalgia and other conditions

Some practitioners consider IV vitamin C for autism because of its: 

• Antioxidant effects – reducing oxidative stress, which is elevated in many children with autism.
• Anti-inflammatory properties – calming neuroinflammation and microglial activation.
• Support for neurotransmitter synthesis – vitamin C is a cofactor in dopamine and norepinephrine production.
• Possible mast cell stabilization – relevant in children with autism and comorbid mast cell activation syndrome (MCAS).
• Histamine degradation support – helps recycle tetrahydrobiopterin (BH4), indirectly involved in histamine metabolism.

 

It has been found that liposomal vitamin C can achieve levels in the blood somewhere in between IV-vitamin C and regular vitamin C by food or supplements. 

High levels of vitamin C can cause side effects such as kidney stones.

Liposomal vitamin C is better tolerated than very high doses of standard vitamin C. It looks like things are likely to start going wrong above 3,000mg a day of liposomal.

Healthy people just need a good diet. If they have a poor diet then take a multivitamin.

Liposomal or IV therapy is only for people with real health issues.

People with MCAS plus autism certainly do have health issues.

Ehlers–Danlos syndrome (and milder subclinical versions) is linked to MCAS, ADHD, autism and Tourette’s. So that is another group to consider.

Fibromyalgia was put forward (by me) as a step towards autism in some females, in subsequent levels of their family tree.

So overall the idea of liposomal vitamin C has much more merit than a natural sceptic would have first thought. (There are loads of YouTube videos of people doing this, and likely many did not really need it.)

 

Whey protein as an antioxidant 

This topic was recently highlighted by our reader Stephen and it naturally fits into this post.

Back in 2013 when I was developing my son’s therapy I had to choose between NAC and whey protein to boost glutathione (GSH), the body’s key antioxidant. I chose NAC.

Here is a great paper to support the use of whey protein.

 

Improving Antioxidant Capacity in Children With Autism: A Randomized, Double-Blind Controlled Study With Cysteine-Rich Whey Protein 

Previous studies indicate that children with autism spectrum disorder (ASD) have lower levels of glutathione. Nutritional interventions aim to increase glutathione levels suggest a positive effect on ASD behaviors, but findings are mixed or non-significant. A commercially available nutritional supplement comprising a cysteine-rich whey protein isolate (CRWP), a potent precursor of glutathione, was previously found to be safe and effective at raising glutathione in several conditions associated with low antioxidant capacity. Therefore, we investigated the effectiveness of a 90-day CRWP intervention in children with ASD and examined whether intracellular reduced and oxidized glutathione improvements correlated with behavioral changes. We enrolled 46 (of 81 screened) 3-5-year-old preschool children with confirmed ASD. Using a double-blind, randomized, placebo-controlled design, we evaluated the effectiveness of daily CRWP (powder form: 0.5 g/kg for children <20 kg or a 10-g dose for those >20 kg), compared with placebo (rice protein mimicking the protein load in the intervention group), on glutathione levels and ASD behaviors assessed using different behavioral scales such as Childhood Autism Rated Scale, Preschool Language Scale, Social Communication Questionnaire, Childhood Behavioral Checklist and the parent-rated Vineland Adaptive Behavior Scale, 2nd edition (VABS-II). Forty children (CRWP, 21; placebo, 19) completed the 90-day treatment period. Improvements observed in some behavioral scales were comparable. However, the VABS-II behavioral assessment, demonstrated significant changes only in children receiving CRWP compared to those observed in the placebo group in the composite score (effect size 0.98; 95% confidence intervals 1.42-4.02; p = 0.03). Further, several VABS-II domain scores such as adaptive behavior (p = 0.03), socialization (p = 0.03), maladaptive behavior (p = 0.04) and internalizing behavior (p = 0.02) also indicated significant changes. Children assigned to the CRWP group showed significant increases in glutathione levels (p = 0.04) compared to those in the placebo group. A subanalysis of the VABS-II scale results comparing responders (>1 SD change from baseline to follow up) and non-responders in the CRWP group identified older age and higher levels of total and reduced glutathione as factors associated with a response. CRWP nutritional intervention in children with ASD significantly improved both glutathione levels and some behaviors associated with ASD. Further studies are needed to confirm these results.

 

This study used a special commercial product called Immunocal, a cysteine-rich whey protein isolate (CRWP) that serves as a potent glutathione precursor.

There are less expensive alternatives to Immunocal that still offer high-quality, undenatured, cysteine-rich whey protein, especially if your goal is to support glutathione production without paying premium prices. These products are typically marketed as cold-processed, non-denatured whey protein concentrates or isolates, and some are even made from the same raw material sources as Immunocal.

If you want to further increase absorption you can even make a liposomal version of a cysteine-rich whey protein!! 

Regular body builders’ whey protein is great to help build muscles and to maintain muscle mass in seniors, but it is not the ideal source of cysteine. It has degraded during the production process, that why there are fancy ones available.

I think Stephen would indeed be well advised to add a scoop of cysteine-rich whey protein isolate (CRWP) to his sons’ diets. It should have a more prolonged effect than NAC. For young children with autism NAC really needs to be given 3-4 times a day.

You can have too much cysteine. You do not need high dose of both NAC and CRWP.

 

Back to liposomal EGCG

If you read the reviews many people find commercial liposomal supplements no more effective than the much cheaper, regular ones. I wonder why. Most likely they were not well formulated, or they degraded by the time they were used. These products are not heat or light stable.

Many manufactured products like fish oil supplements no longer maintain the health benefit of the genuine article (fish, in this case). This is because the product degraded and sometimes can even have a negative behavioral effect. 

 

Many healthy natural products like catechins or curcuma have very low bioavailability

There is a long list of healthy products that should be therapeutic in autism including:

·        Green tea catechins like EGCG

·        Turmeric/Curcuma

·        Resveratrol

·        Cocoa

·        Many herbs (sage, oregano, rosemary, Bacopa monnieri, ginseng, lions mane, etc)

They generally have very low bioavailability and so they work great in the lab, but much less so in humans; unless you consume very large amounts, for example turmeric in an Indian diet.

 

EGCG

I have written about EGCG in the past and have highlighted the research from Spain, more specifically from the beautiful city of Barcelona (just avoid visiting during the peak summer months). The research showed a benefit in Fragile X and Rett syndrome. As usual, no customized intervention has yet been brought to the market.

https://www.epiphanyasd.com/search/label/EGCG?max-results=20

Yet another study showing the potential benefit of EGCG, was published recently, this time in Pakistan.

 

Cross-linking catechins with neuro-regulatory model for autism spectrum disorder: A management in rats’ experiment 

We found that BDNF levels returned to normal levels within the groups who received Catechins treatment at III, IV, and V concentrations (compared to Group II), showing Catechins could potentially treat autism-like symptoms. The BDNF values measured in nano-grams per millilitre were Group I (13.1±0.3), followed by Group II (5.1±0.2) and Group III (9.8±0.3), Group IV (8.0±0.3), and then Group V (10.1±0.3). The BDNF concentration measured in Groups III, IV and V surpassed the BDNF level of Group II (PPA-induced) per results from a post-hoc Tukey's test at p 

Catechins successfully decreased neuroinflammatory markers throughout the brain and establish protective brain mechanisms that potentially improve ASD-associated behavioral symptoms. Rats given 100, 200, and 400 mg/kg of various catechins showed increases in BDNF levels of up to 75%, 61%, and 77%, respectively, as opposed to only 39% for rats that received no treatment. The findings of a study suggested a continuous and expandable neuroprotective effect based on dose strength. The experimental results demonstrated that in ASD models, catechins offer a potent and dosage-dependent defense against neuroinflammatory injuries.

  

This study confirms that epigallocatechin gallate (EGCG), among catechins, shows great promise for managing neuroinflammation in ASD patients. The results indicate that catechins deliver substantial reductions in neuroinflammatory markers, as they serve as protective element that improves behavioral and cognitive manifestations of ASD. Future investigations must explore mechanisms of effect and find best-use dosages for catechins while establishing their safety and lasting effect durations.

 

Then I came across this paper where the university made their own liposomal version of EGCG and tried it on their model of Parkinsons’ disease. It also worked very well. Autism is not Parkinsons’ but both conditions feature activated microglia, the brain’s immune cells that are also tasked with synaptic pruning housekeeping duties.

 

Epigallocatechin-3-Gallate-Loaded Liposomes Favor Anti-Inflammation of Microglia Cells and Promote Neuroprotection

Microglia-mediated neuroinflammation is recognized to mainly contribute to the progression of neurodegenerative diseases. Epigallocatechin-3-gallate (EGCG), known as a natural antioxidant in green tea, can inhibit microglia-mediated inflammation and protect neurons but has disadvantages such as high instability and low bioavailability. We developed an EGCG liposomal formulation to improve its bioavailability and evaluated the neuroprotective activity in in vitro and in vivo neuroinflammation models. EGCG-loaded liposomes have been prepared from phosphatidylcholine (PC) or phosphatidylserine (PS) coated with or without vitamin E (VE) by hydration and membrane extrusion method. The anti-inflammatory effect has been evaluated against lipopolysaccharide (LPS)-induced BV-2 microglial cells activation and the inflammation in the substantia nigra of Sprague Dawley rats. In the cellular inflammation model, murine BV-2 microglial cells changed their morphology from normal spheroid to activated spindle shape after 24 h of induction of LPS. In the in vitro free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, EGCG scavenged 80% of DPPH within 3 min. EGCG-loaded liposomes could be phagocytized by BV-2 cells after 1 h of cell culture from cell uptake experiments. EGCG-loaded liposomes improved the production of BV-2 microglia-derived nitric oxide and TNF-α following LPS. In the in vivo Parkinsonian syndrome rat model, simultaneous intra-nigral injection of EGCG-loaded liposomes attenuated LPS-induced pro-inflammatory cytokines and restored motor impairment. We demonstrated that EGCG-loaded liposomes exert a neuroprotective effect by modulating microglia activation. EGCG extracted from green tea and loaded liposomes could be a valuable candidate for disease-modifying therapy for Parkinson’s disease (PD).

 

Looks great, but you cannot buy their product. It then appeared that people are already making liposomal supplements at home.

Dig a little deeper to see what other clever ideas exist in the university research world that might make DIY versions better. 

 

Liposomal Formulations for an Efficient Encapsulation of Epigallocatechin-3-Gallate: An In-Silico/Experimental Approach

As a part of research project aimed to optimize antioxidant delivery, here we studied the influence of both salts and lipid matrix composition on the interaction of epigallocatechin-3-gallate (EGCG) with bilayer leaflets. Thus, we combined in silico and experimental methods to study the ability of neutral and anionic vesicles to encapsulate EGCG in the presence of Ca2+ and Mg2+ divalent salts. Experimental and in silico results show a very high correlation, thus confirming the efficiency of the developed methodology. In particular, we found out that the presence of calcium ions hinders the insertion of EGCG in the liposome bilayer in both neutral and anionic systems. On the contrary, the presence of MgCl2 improves the insertion degree of EGCG molecules respect to the liposomes without divalent salts. The best and most efficient salt concentration is that corresponding to a 5:1 molar ratio between Mg2+ and EGCG, in both neutral and anionic vesicles. Concerning the lipid matrix composition, the anionic one results in better promotion of the catechin insertion within the bilayer since experimentally we achieved 100% EGCG encapsulation in the lipid carrier in the presence of a 5:1 molar ratio of magnesium. Thus, the combination of this anionic liposomal formulation with magnesium chloride, avoids time-consuming separation steps of unentrapped active principle and appears particularly suitable for EGCG delivery applications.

 

The Mozafari method for Liposomal delivery

The latest methods used in universities to make liposomal products cannot be entirely replicated at home, but there is a well-known method developed by Dr Mohammad Mozafari that has been proved to increase bioavailability 2 to 8 times. The Mozafari method is used today by biohackers at home. Often they seem to skip some important steps.

We can fine tune his method, for example by noting the research showing that magnesium ions can help stabilize the liposomes and improve encapsulation of EGCG. Calcium ions have a very negative effect and so make sure no calcium (for example, from hard water) enters the process. YouTubers just use tap water. So use high-quality deionized (DI) water and add Magnesium Chloride (MgCl₂).

Anionic liposomes (negatively charged phospholipids) promote better EGCG insertion compared to neutral liposomes. With Mg²⁺, anionic liposomes reached 100% encapsulation efficiency experimentally. So it was actually perfect.

Magnesium chloride (MgCl₂) at about a 5:1 molar ratio relative to EGCG

(Example: for 500 mg EGCG ≈ 1.1 mmol, add ~5.5 mmol MgCl₂ — roughly 670 mg MgCl₂·6H₂O)

Both pH and temperature control are important and seem to get ignored by YouTubers.

Choose the right lipid. Here are the choices:

Most DIYers are using Lecithin (sunflower or soy), which contains phosphatidylcholine (PC), plus other substances you do not want. It is cheaper than pure PC.

If you are making liposomal vitamin C, glutathione, DHA or EGCG for therapeutic use (e.g., autism, MCAS, oxidative stress), pure PC gives superior performance.

Lecithin is zwitterionic, meaning it contains both positive and negative charges, but is overall electrically neutral. This dual nature is what makes lecithin perfect for encapsulating both water-soluble (like vitamin C) and fat-soluble (like curcumin) compounds in liposomes.

For closer to University-grade work we need to look at pure chemicals.

·        Phosphatidylcholine (PC) — neutral

·        Phosphatidylserine (PS) — anionic (negative charged)

·        CHEMS (Cholesteryl Hemisuccinate), a negatively charged cholesterol derivative.

·        Cholesterol

 

Component             Role  

PC                               Bilayer structure & fluidity         

PS                               Anionic charge, Mg²⁺ interaction          

Cholesterol               Stabilization (optional)    

CHEMS                      Additional anionic charge (optional)

 

Phosphatidylserine (PS) is itself therapeutic

PS naturally concentrates in the brain, especially in neuronal membranes.

It is known to support memory, attention, synaptic function, and neuroplasticity — ideal for neurodegenerative and developmental conditions.

PS is negatively charged (anionic), which helps form stable liposomes and can improve encapsulation of positively charged or hydrophilic molecules like EGCG.

PS has functional activity, beyond just being a carrier, PS itself may synergize with EGCG and other cognitive-enhancing compounds.

Adding cholesterol makes the liposome less leaky and more resistant to degradation. Without cholesterol, liposomes are more prone to oxidation, fusion, or breakdown over time

  

Example for 2 g Total Lipids:

Lipid Component	Weight (grams)	Percentage
PC	1.2 g	60%
PS	0.4 g	20%
Cholesterol	0.4 g	20%

 

• PC provides a stable bilayer and good liposome formation.
• PS introduces a negative charge that enhances electrostatic interaction with Mg²⁺ and EGCG.
• Cholesterol improves membrane rigidity and stability, helping prevent leakage.
• You can adjust cholesterol slightly depending on how rigid you want the membrane.
• Maintain MgCl₂ at ~5:1 molar ratio to EGCG in the aqueous buffer for optimal encapsulation, as per references.

EGCG is highly oxidation-sensitive.

Both vitamin C (ascorbic acid) and vitamin E (tocopherol) protect:

·        the lipids in the liposome from peroxidation,

·        the EGCG itself from degradation.

So it is wise to add both vitamin C and E.

• Vitamin E is lipid-soluble and embeds in the bilayer.
• Vitamin C is water-soluble and protects the aqueous core.

 

Here is the home version.

 

 

Equipment

• Glass beaker or jar
• Ultrasonic cleaner (sold to clean jewellery)
• Stirring rod
• pH strips or meter
• Dark glass storage bottle

 

Method

1.     Mix Vitamin E with PS

o    Combine PS powder and vitamin E oil or powder thoroughly in a small container.

2.     Prepare aqueous phase

o    Dissolve EGCG powder and magnesium chloride in ~20 mL PBS or distilled water with buffer salts.

o    Add vitamin C to this aqueous solution last and stir gently until dissolved.

3.     Hydrate lipids

o    Slowly add the aqueous phase (EGCG + MgCl₂ + vitamin C) to the PS + vitamin E mix.

o    Stir or vortex gently to disperse.

4.     Sonicate

o    Place the mixture in an ultrasonic cleaner bath for 20–30 minutes, stirring occasionally.

o    Solution should become milky/opalescent, indicating liposome formation.

 

How to Use Ultrasonic Cleaner for Liposomal EGCG

1.     Prepare your liposome suspension in a suitable sealed container—usually a small glass vial or bottle with a tight lid (e.g., amber glass bottle or glass vial).

2.     Fill the ultrasonic cleaner tank with clean water—enough so that when you place your container in it, the water level reaches just below the lid or about 2/3 up the container’s height. The water must not overflow into your liposome container

3.     Place your sealed bottle/vial into the ultrasonic bath, making sure it sits upright and stable.

4.     Turn on the ultrasonic cleaner for the recommended at medium power.

5.     During the process, keep an eye on the temperature—if the water or sample gets too warm (>40°C), pause and let it cool, since heat will degrade EGCG.

6.     After sonication, remove the bottle and store the liposomal EGCG in a dark, refrigerated place.

 

Important Tips

• Use sealed containers to avoid contamination or water ingress.
• Never put the liposomal suspension directly into the ultrasonic cleaner’s water bath.
• If your ultrasonic cleaner has a temperature control or timer, use those settings to protect the sample.
• Clean the ultrasonic tank well before and after use.

The final product will be stable for 7 days in the fridge.

You can freeze portion sized doses in a silicone ice cube tray. Later store in the freezer in a zip lock bag for 2-3 months. Defrost in the fridge, one by one, as you need it.

Keeping the temperature below 40°C is essential when sonifying delicate compounds like EGCG, vitamin C, and phospholipids (especially phosphatidylserine). They degrade or oxidize easily when exposed to excessive heat.

 

 1. Use a Cold Water Bath

• Fill the ultrasonic cleaner with cold water (4–10°C).
• Add ice cubes to keep it cold.
• Replenish ice as needed during sonication.

 

2. Monitor Temperature

• Use a probe thermometer or an infrared laser thermometer.
• Check the temperature of your sample, not just the water bath.

 

Ultrasonic waves create cavitation — rapid formation and collapse of microbubbles — which:

• Generates localized heat (tiny hot spots in the solution)
• Transfers energy into the liquid, raising the overall temperature gradually
• Can increase your solution temperature from room temp to 50–60°C in 10–15 minutes if not managed

Even if the water bath feels lukewarm, the inside of your beaker can be much hotter, especially in the center.

 

 Sensitive Ingredients at Risk

• EGCG starts to degrade above ~40°C
• Phospholipids can oxidize or become unstable
• Vitamin C rapidly degrades in heat and light

 

Best Practices to Avoid Overheating

Tip	Why
Use an ice-cold bath	Keeps temp under control during sonication
Sonicate in short bursts (30s ON, 30s OFF)	Prevents buildup of heat
Stir gently before and after sonication	Helps distribute heat and emulsify
Monitor temperature regularly	Keeps you in the safe zone (under 40°C)
Work with small batches (≤100 mL)	Easier to cool and control

 

Sonication is the process of using high-frequency sound waves—usually ultrasound—to agitate particles in a liquid. In making liposomes, sonication helps break down large lipid aggregates into smaller, uniform liposomes by creating tiny bubbles that collapse and generate energy, mixing and dispersing the components more thoroughly.

In simpler terms, sonication is like using sound waves to gently shake and break up the mixture so that the fat-like particles (liposomes) form smaller, more consistent droplets, which can improve how well the active ingredient (like EGCG) gets absorbed by your body.

  

Conclusion

Immunocal, a cysteine-rich whey protein isolate (CRWP) does show a benefit in autism. It is an OTC product that is sold online.

EGCG is the most abundant catechin in green tea.  When taken orally, EGCG has poor absorption even at daily intake equivalent to 12 cups of green tea.

EGCG does show merit in autism research studies, but to be effective in humans the bioavailability needs to be improved. Using liposomes to encase the EGCG is a promising approach.

Will the method proposed in this post be effective?

I asked Gemini AI to comment on the method and it replied: 

"The EGCG method you described is a fantastic example of a DIY approach that is grounded in sound scientific principles. By using specific lipids, optimizing the formulation with magnesium, and strictly controlling the process, it goes far beyond the simplistic and often ineffective methods seen online.

While it won't produce a pharmaceutical-grade product, it is highly likely to create a solution that contains a significant number of genuine, stable, and correctly sized liposomes. This makes it far more likely to be an effective delivery system than the typical DIY liposomal vitamin C, which is often just an unverified emulsion."

Some people do grow their own broccoli sprouts to make sulforaphane, others grow wheat sprouts for spermidine. Some people grow their own probiotic bacteria. Making tumeric balls is a simple way to get the benefits of tumeric. There are many home-made options, and I think the parent almost certainly benefits. 

You would think that some enterprising pharmacist in Barcelona would start producing small batches of liposomal EGCG, using research grade equipment. I think Rett syndrome and Fragile X syndrome parents would buy it. Not to mention those who have parents diagnosed with Parkinson's or Alzheimer's.

   

  

Will Anavex for “Autisms” be worth the wait and the price, compared to Russian OTC Afobazole?

US-Russia cooperation has long been possible in Space, but not so often in Medicine. NASA reportedly pays Russia $85 million per astronaut to go the International Space Station (ISS).  The US Space Shuttle program ended in 2011, leaving a Russian Soyuz rocket the only way to the ISS.

This post comes ahead of the dietary autism post, awaited by Tanya.  It really is just a brief follow-on from the previous post. I have only just come across Anavex, which does add weight to the first post on sigma-1R.
                                                                                                               

Hundreds of millions of dollars are being spent in the US to develop a safe sigma-1R agonist (Anavex 2-73). This drug is being trialed in various autisms (Rett, Fragile X and Angelman syndromes), Parkinson’s and Alzheimer’s.

In the last post I wrote about a cheap OTC anxiety drug from Russia, called Afobazole, that appears to be a safe sigma-1R agonist.  This drug has also recently been trialed in autism and Parkinson’s - the same targets as Anavex.

I did make the point in my original sigma-1 post that I am interested in existing therapies, rather than potential ones, so I did not include Anavex, or any other research drug, in that post. Anavex is nonetheless interesting, because their research studies further support the suggestion that targeting ER stress via sigma-1 is an interesting avenue to pursue.  

ERStress and Protein Misfolding in Autism (and IP3R again) and perhaps what to do about it - Activation of Sigma-1 Chaperone Activity by Afobazole?

Anavex is claiming precision medicine, but in fact sigma-1R agonists appear more like the opposite, at least in terms of who you target.  The majority of both common and rare neurological disorders look like they should benefit from reducing ER Stress (from whatever cause); it is a shared feature.  So it looks more like a shotgun approach; that is actually a good thing, if it were to drive the price down.

What is needed is an affordable, effective, mass market drug; not an ultra expensive pill just for Rett Syndrome and perhaps a different colour version for Angelman's Syndrome.

Which will prove effective - Anavex or Afobazole? Or perhaps neither.

Having already made the case for Soyuz in my earlier post, here is the case for NASA, and for those with NASA-sized budgets, courtesy of  https://www.anavex.com/

Animal Study Details Motor, Sensory and Respiratory Benefits of Anavex 2-73, Supporting Phase 2 Trials

Treatment with Anavex 2-73 was seen to improve motor skills, acoustic responses and visual acuity in a mouse model of Rett syndrome, supporting ongoing Phase 2 studies in patients.

Its use also helped to lessen abnormal movements and ease breathing in these mice, its researchers said.

The study, “ANAVEX®2-73 (blarcamesine), a Sigma-1 receptor agonist, ameliorates neurologic impairments in a mouse model of Rett syndrome,” was published in the journal Pharmacology Biochemistry and Behavior.

Anavex 2-73 (blarcamesine) is an oral investigational therapy developed by Anavex Life Sciences that works by activating the sigma-1 receptor (S1R), a protein involved in the correct folding of other proteins.

S1R activation results in reduced toxic accumulation of misfolded proteins, as well as lesser dysfunction in mitochondria (a cell’s “powerhouse”), oxidative stress and neuroinflammation, all involved in Rett syndrome. (Oxidative stress is an imbalance between the production of free radicals — potentially harmful molecules associated with a number of diseases — and the generation of antioxidant defenses.)

Researchers at Anavex, assisted by PsychoGenics, evaluated the potential treatment’s specific effects on Rett symptoms in a validated mouse model.

They assessed motor function (balance, motor coordination, locomotion, and abnormal movements or stereotypies), sensory function (reflex responses to sound stimuli and visual clarity), and respiratory function.

Motor and sensory functions were assessed in younger mice, while visual acuity and breathing were measured in older animals.

Results showed that Anavex 2-73 significantly eased motor dysfunction, and deficits in acoustic and visual responses compared to mice given a placebo.

Anavex 2-73 also induced a significant reduction in two distinctive features of Rett syndrome found in these mice: hind-limb clasping (an abnormal posture comparable to hand stereotypies in people with Rett), and apnea (involuntary breath-holding) that is the most concerning breathing abnormality in Rett syndrome, the researchers said. These improvements were mainly dependent on treatment dose and duration.

“In conclusion, the data demonstrate that [Anavex 2-73] is effective in ameliorating multiple neurobehavioral phenotypes in [Rett] mice,” the researchers wrote. “In line with previous animal and human studies [in other neurodegenerative diseases], [Anavex 2-73] also showed a good safety profile,” they added.

These data served as a proof-of-concept for an ongoing safety and efficacy Phase 2 trial called RS-001 (NCT03758924, still enrolling) in the U.S., and for the Phase 2 AVATAR study (NCT03941444) in Australia. These trials together will evaluate Anavex 2-73 in up to 51 women with Rett syndrome.

Conclusion

It may be that Anavex is far superior to the cheap Afobazole. Like the space shuttle was far more advanced than the Soyuz. 

But what if the cheap Afobazole is quite good enough?  Like the cramped, but reliable Soyuz rocket.

Anavex/Afobazole will not cure any severe neurological condition, just improve it, so it will need to be part of a polytherapy. That means the patient will need to be able to afford multiple drugs, somehow.

Coming back to those autisms, what if your daughter has Rett Syndrome, or son has Fragile-X Syndrome ?  Wait a few years for Anavex and for someone else to pay for it? or make do with some cheap Afobazole?

ER Stress and Protein Misfolding in Autism (and IP3R again) and perhaps what to do about it - Activation of Sigma-1 Chaperone Activity by Afobazole?

Today’s post may require even regular readers to refresh their memories and look up the meaning of some words.

There really is a lot in this post. I had to read it twice.

As is often the case, this post started at the end with the therapy (a trial of Afobazole) and then I just looked at why it might be effective.

Activate Chaparones

Today's post is all about sigma-1 receptors and the many clever things that happen when they are activated.

Even the above diagram showing the effect of Sigma-1R is incomplete!

In the mouse study below, the Russian researchers looked at the effect of Afobazole treatment just over a few days; I think other effects might have developed if they had looked at an extended time period. They focus on Sigma1-R receptors modulating NMDA-based neurotransmission, but there seem to many possible further effects within the Endoplasmic Reticulum that relate specifically to autism. These researchers have published other studies using Afobazole, including recently one on Parkinson's disease. 

Afobazole Alleviates Cognitive Rigidity in Experimental Model of Autism Spectrum Disorders.

The multifactorial nature of ASD precludes the use of its modern genetic models in the study of pharmacologic effects exerted on entire symptomatic complex of autism although they could relate functional correction of ASD with a certain gene. In experiments, the models of idiopathic ASD are based on inbred mice selected by behavioral phenotype. BALB/c mice demonstrate pronounced autism-relevant behavioral phenotype characterized by low level of social relations, high levels of anxiety and aggression, increased brain weight, undeveloped corpus callosum, and lower serotonin concentration in the brain [7,12]. The emotional stress reaction (ESR) in these animals is associated with weaker binding capacity of the benzodiazepine site in GABAA receptor [6]. Transformation of ESR into the cell stress augments reception in the domain responsible for binding the endo- and exogenous ligands of sigma 1 receptor chaperon protein (Sigma1R) [1] responsible for adaptive reactions [8]. In addition, Sigma1R stimulate BDNF and NGF synthesis, promote the growth and arborization of nerve terminals, and control functional activity of potassium, calcium, and chloride ion channels and a variety of neuroreceptors [5,8,13]. Thus, this chaperon protein can be an important player in physiological and pharmacological regulation of ASD features.

Afobazole is a non-benzodiazepine anxiolytic drug that acts via activation of Sigma1R and interaction with MT1 and MT3 melatonin receptors and a regulatory site of MAO-A [4]. Clinical observations showed that Afobazole optimizes psychophysiological parameters in emotionally unstable persons without impairing attention, psychomotor responsiveness, and decision-making alertness in the model of operator work. The drug is characterized by mild activation effect and reduces anxiety, thus promoting adaptation to novel environment [2]. This work was designed to examine the effects of Afobazole on cognitive rigidity in BALB/c mice.

Evidently, enhanced motor activity of Afobazole treated BALB/c mice reflected the anxiolytic effect of this drug, which stimulated exploratory behavior aimed at solving the novel task. Thus, Afobazole improved adaptation to changing environment

The present study revealed the potency of Afobazole to promote retraining and reversal learning of BALB/c mice, which manifested in increased rate of adaptation to novel environment and more effective solution of the modified task. Afobazole interacts with Sigma1R receptors and induces their activation [1]. It cannot be excluded that the anxiolytic effect of Afobazole is accompanied by up-regulation of Sigma1R chaperone functions, because this drug normalizes the stress-induced down-regulation of reception in benzodiazepine site of GABAA receptor [6]. A large cluster of Sigma1R receptor was revealed in the hippocampus that plays a key role in adaptive behavior related to building of spatial cognitive maps, learning, and memory. Sigma1R receptors modulate NMDA-based neurotransmission; they can enhance spontaneous release of glutamate in the hippocampus, potentiate glutamate-induced release of neurotrophic factor, and participate in synaptic plasticity [8]. However, Sigma1R receptors regulate cognitive processes under disturbed neurotransmitter balance only. All these data agree with our previous findings and with the current views on the mechanism of Afobazole action [1,4,5]. Thus, the mode of action and pharmacological effects of Afobazole are promising features, which justify the hopes to use it as an effective remedy to treat cognitive rigidity in ASD patients

More on sigma-1 and NMDA receptors:-

NMDA Receptors Are Upregulated and Trafficked to the Plasma Membrane after Sigma-1 Receptor Activation in the Rat Hippocampus

Sigma-1 receptors (σ-1Rs) are endoplasmic reticulum resident chaperone proteins implicated in many physiological and pathological processes in the CNS. A striking feature of σ-1Rs is their ability to interact and modulate a large number of voltage- and ligand-gated ion channels at the plasma membrane. We have reported previously that agonists for σ-1Rs potentiate NMDA receptor (NMDAR) currents, although the mechanism by which this occurs is still unclear. In this study, we show that in vivo administration of the selective σ-1R agonists (+)-SKF 10,047 [2S-(2α,6α,11R*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol hydrochloride (N-allylnormetazocine) hydrochloride], PRE-084 (2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate hydrochloride), and (+)-pentazocine increases the expression of GluN2A and GluN2B subunits, as well as postsynaptic density protein 95 in the rat hippocampus. We also demonstrate that σ-1R activation leads to an increased interaction between GluN2 subunits and σ-1Rs and mediates trafficking of NMDARs to the cell surface. These results suggest that σ-1R may play an important role in NMDAR-mediated functions, such as learning and memory. It also opens new avenues for additional studies into a multitude of pathological conditions in which NMDARs are involved, including schizophrenia, dementia, and stroke.

Afobazole is primarily used to treat mild anxiety.  Indeed it appears that sigma-1 receptor activation ameliorates anxiety through NR2A-CREB-BDNF signalling.  NR2A is a sub-unit of NMDA receptors.

Sigma-1 receptor activation ameliorates anxiety-like behavior through NR2A-CREB-BDNF signaling pathway in a rat model submitted to single-prolonged stress.

It does seem that activating the sigma-1 receptor might be another of those nexuses in treatment, where different dysfunctions in autism might well respond to the same therapy.  Recall how many functions of the Endoplasmic Reticulum are impaired in autism, such as the all important calcium homeostasis. 

It also might account for some of the people with autism that respond to Memantine/Nameda and Donepezil. My old post on IP3R and the endoplasmic reticulum, looked at the interesting hypothesis proposed by Gargus.

Is dysregulated IP3R calcium signaling a nexus where genes altered in ASD converge to exert their deleterious effect?

Components of a typical animal cell:

1.                 Nucleolus

2.                 Nucleus

3.                 Ribosome (little dots)

4.                Vesicle

5.                Rough endoplasmic reticulum

6.                Golgi apparatus (or "Golgi body")

7.                Cytoskeleton

8.               Smooth endoplasmic reticulum

9.               Mitochondrion

10.            Vacuole

11.            Cytosol (fluid that contains organelles)

12.             Lysosome

13.             Centrosome

14.             Cell membrane

Endoplasmic Reticulum (ER) and ER Stress

The endoplasmic reticulum (ER) is the cellular organelle in which protein folding, calcium homeostasis, and lipid biosynthesis occur. Stimuli such as oxidative stress, ischemic insult, disturbances in calcium homeostasis, and enhanced expression of normal and/or folding-defective proteins lead to the accumulation of unfolded proteins, a condition referred to as ER stress.

Prolonged ER stress typically results in cell death by apoptosis; an answer to “where did all the Purkinje cells go?”, in people with severe autism, perhaps.  

ER stress is known to affect "neurite outgrowth", which is all the bits like dendrites. Purkinje cells have the most dendrites.  Loss of Purkinje cells affects your motor skills and the Pukinje cell layer is found to be severely depleted in people with autism. Many people with autism, even some Aspies, have poor motor skills. 

Research shows that exercise suppresses Purkinje cell losss and that the ones remaining in autistic brains are likley dysfunctional. When synaptic pruning works correctly each Purkinje cell in an adult receives only one climbing fiber input, in ASD models there is an abundance of climbing fibers. It does seem that with enough practice you may overcome poor motor skills in autism.

Interestingly, in the research we see that both Atorvastatin and Rosuvastatin enhance neurite outgrowth. Atorvastatin has long been part of my PolyPill for severe autism.

For effective synaptic pruning you need microglia that are not activated, so shift them back to M0.  This is another part of my PolyPill.

Protein folding is the physical process by which a protein chain acquires its native 3-dimensional structure, a conformation that is usually biologically functional.

Molecular chaperones are a class of proteins that aid in the correct folding of other proteins, sigma-1 is one example.

A protein is considered to be misfolded if it cannot achieve its normal native state and function.

Incorrect protein folding is a common feature of neurodegenerative disease.

An emerging approach is to use pharmaceutical chaperones to fold mutated proteins to render them functional.

As will be seen in the research, ER stress is a feature of severe autism and indeed schizophrenia.

The result is that perfect genes do not produce perfect functional proteins.  They produce misfolded perfect proteins that cannot function.

Misfolded proteins can interact with one another and form structured aggregates and gain toxicity through intermolecular interactions, but that would lead to a degenerative brain disease (Alzheimer’s, Huntington’s, Parkinson’s etc).  So, the misfolding in autism, if present, it not catastrophic (except perhaps for those Purkinje cells); but a nice folded shirt does give a better result than a crumpled one. Better keep your proteins neatly folded. 

Is there ER Stress in Autism?

The short answer is yes, at least in the kind of autism that leads to young human brains  being donated to medical research.  

Autism research based on human brain tissue is biased towards severe autism (they can die in childhood), whereas many/most clinical trials are now biased towards mild autism (having participants who are fully verbal and cooperative makes life easier for researchers, but their young brains do not get donated to medical research).   

Altered Expression of Endoplasmic Reticulum Stress-Related Genes in the Middle Frontal Cortex of Subjects with Autism Spectrum Disorder

The endoplasmic reticulum (ER) is an important organelle responsible for the folding and sorting of proteins. Disturbances in ER homeostasis can trigger a cellular response known as the unfolded protein response, leading to accumulation of unfolded or misfolded proteins in the ER lumen called ER stress. A number of recent studies suggest that mutations in autism spectrum disorder (ASD)-susceptible synaptic genes induce ER stress. However, it is not known whether ER stress-related genes are altered in the brain of ASD subjects. In the present study, we investigated the mRNA expression of ER stress-related genes (ATF4, ATF6, PERK, XBP1, sXBP1, CHOP, and IRE1) in the postmortem middle frontal gyrus of ASD and control subjects. RT-PCR analysis showed significant increases in the mRNA levels of ATF4, ATF6, PERK, XBP1, CHOP, and IRE1 in the middle frontal gyrus of ASD subjects. In addition, we found a significant positive association of mRNA levels of ER stress genes with the diagnostic score for stereotyped behavior in ASD subjects. These results, for the first time, provide the evidence of the dysregulation of ER stress genes in the brain of subjects with ASD.

Increase in mRNA levels of endoplasmic reticulum stress genes in the middle frontal gyrus of autism spectrum disorder (ASD) subjects. mRNA levels of endoplasmic reticulum stress genes were determined by qRT-PCR in the middle frontal gyrus of ASD (n = 13) and control (n = 12) subjects. The Ct values were normalized to the mean of 18S and β-actin. a Activating transcription factor 4 (ATF4). b Activating transcription factor 6 (ATF6). c Protein kinase-like endoplasmic reticulum kinase (PERK). d X-box protein 1 (XBP1). e Spliced X-box protein 1 (sXBP1). f CCAAT-enhancer-binding protein homologous protein (CHOP). g Inositol-requiring enzyme 1 (IRE1). * p < 0.05, ** p < 0.01, and *** p < 0.0001 vs. controls.

We found significant increases in ATF4, ATF6, PERK, XBP1, CHOP, and IRE1 mRNA levels in the middle frontal gyrus of ASD subjects. Among these molecules, CHOP is known to interact with the heterodimeric receptors GABA_B1aR/GABA_B2R and inhibits the formation of heterodimeric complexes resulting in the intracellular accumulation and reduced cell surface expression of receptors [34]. Interestingly, decreased levels of GABA_B1R and GABA_B2R have been found in the brain of ASD subjects [35]. What are the downstream mechanism mediating ER stress-induced changes in central nervous system function? One potential mechanism is inflammation. Accumulating evidence suggest that pathways activated by the ER stress response induce inflammation. When activated, all three sensors of the UPR, PERK, IRE1, and ATF6, participate in upregulating inflammatory processes. It is known that PERK and IRE1 activation can interfere with NFκB inhibitory signals, thereby promoting a proinflammatory response [36]. In addition, CHOP has been shown to induce the expression of proinflammatory cytokines such as IL-23 [37]. Moreover, ER stress activates NLRP3 inflammasomes via thioredoxin-interacting protein (TXNIP), leading to increases in proinflammatory cytokine levels [38,39]. In this regard, our earlier studies using the same tissue samples of the present study found increased levels of proinflammatory cytokines IL-1β and IFN-γ in the middle frontal gyrus of ASD subjects [30].

Also, chronic ER stress is known to induce cellular apoptosis through a number of pathways including CHOP, calcium signaling, and microRNAs [40]. Activation of PERK triggers a series of transcriptional responses mediated by ATF4 and CHOP, which in turn inhibit the expression of anti-apoptotic protein Bcl2 and induce pro-apoptotic proteins such as Bcl2-interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA) [40]. The induction of pro-apoptotic signaling pathway results in the activation of BAX- and BAK-dependent apoptosis at the mitochondria and the activation of the caspase cascade [41]. Interestingly, decrease in Bcl2, but increase in p53 protein levels have been reported in the frontal cortex of ASD subjects [42].

We found that mRNA levels of ER stress genes are positively associated with the stereotyped behavior domain of the ADI-R. It has been shown that autism-associated mutations in NLGN3, which is known to induce ER stress, also increase stereotyped behavior in mice [43]. Similarly, mice lacking CNTNAP2 showed increased repetitive behaviors such as grooming and digging [44], further suggesting that abnormalities in ASD candidate genes implicated in ER stress induce stereotyped behavior in rodents. The present data was collected in a relatively small number of study subjects, which needs further investigation using larger samples before a conclusion can be drawn. Also, the change in gene expression as part of ER stress axis in ASD could be associated with other priming factors functional on different coordinates of this complex neurodevelopmental disorder. Additional studies are warranted to analyze the ER stress-inducing factors with direct relationship to the pathophysiological changes associated with ASD. To further establish a definitive role of ER stress in ASD pathophysiology, the following questions still need to be addressed: (1) Is ER stress in ASD of neurodevelopmental origin? (2) Are there factors other than mutant synaptic proteins that can trigger ER stress leading to ASD phenotype? (3) Is inflammation triggering ER stress or is ER stress triggering inflammation leading to ASD phenotype? (4) Does ER stress induce changes in neural connectivity between key brain regions implicated in ASD pathophysiology? Future studies addressing the above questions might lead to a better understanding of the pathophysiology and provide new avenues of treatment of this disorder.

Cellular stress and apoptosis contribute to the pathogenesis ofautism spectrum disorder

 

Lay Summary

Autism results in significant morbidity and mortality in children. The functional and molecular changes in the autistic brains are unclear. The present study utilized autistic brain tissues from the National Institute of Child Health and Human Development's Brain Tissue Bank for the analysis of cellular and molecular changes in autistic brains. Three key brain regions, the hippocampus, the cerebellum, and the frontal cortex, in six cases of autistic brains and six cases of non‐autistic brains from 6 to 16 years old deceased children, were analyzed. The current study investigated the possible roles of endoplasmic reticulum (ER) stress, oxidative stress, and apoptosis as molecular mechanisms underlying autism. The activation of three signals of ER stress (protein kinase R‐like endoplasmic reticulum kinase, activating transcription factor 6, inositol‐requiring enzyme 1 alpha) varies in different regions. The occurrence of ER stress leads to apoptosis in autistic brains. ER stress may result from oxidative stress because of elevated levels of the oxidative stress markers: 4‐Hydroxynonenal and nitrotyrosine‐modified proteins in autistic brains. These findings suggest that cellular stress and apoptosis may contribute to the autistic phenotype. Pharmaceuticals and/or dietary supplements, which can alleviate ER stress, oxidative stress and apoptosis, may be effective in ameliorating adverse phenotypes associated with autism.

 

Figure 1. Immunoblot analysis of endoplasmic reticulum (ER) stress signals in the autistic cerebellum. Immunoblot analysis of the cerebellum homogenate was performed using p-IRE1a, p-PERK, and total ATF6 antibodies.

  

In summary, we showed the elevation of ER stress signals, oxidative stress, and apoptosis in three regions of autistic brains. Based on these findings, we reason that increased cellular stress and apoptosis in the autistic brain may be associated with the pathogenesis of autism. Because autism is affected by multiple genetic and environmental factors that are case-specific and there are inherent limitations in the postmortem brain, the present observations will need further confirmation in future studies. Further research with larger sample sizes is needed to investigate the association of cellular stress and apoptotic events with the severity and clinical phenotypes of autism.

 

Chaperone Sigma1R mediates the neuroprotective action of afobazole in the 6-OHDA model of Parkinson’s disease

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disease with limited treatment options. Therefore, the identification of therapeutic targets is urgently needed. Previous studies have shown that the ligand activation of the sigma-1 chaperone (Sigma1R) promotes neuroprotection. The multitarget drug afobazole (5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride) was shown to interact with Sigma1Rs and prevent decreases in striatal dopamine in the 6-hydroxydopamine (6-OHDA)-induced parkinsonism model. The aim of the present study was to elucidate the role of Sigma1Rs in afobazole pharmacological activity. Using ICR mice we found that administration of afobazole (2.5 mg/kg, i.p.) or selective agonist of Sigma1R PRE-084 (1.0 mg/kg, i.p.) over 14 days normalizes motor disfunction and prevents decreases in dopamine in the 6-OHDA-lesioned striatum. Afobazole administration also prevents the loss of TH + neurons in the substantia nigra. The pre-administration of selective Sigma1R antagonist BD-1047 (3.0 mg/kg, i.p.) abolishes the activity of either afobazole or PRE-084, as determined using the rotarod test and the analysis of striatal dopamine content. The current study demonstrates the contribution of Sigma1Rs in the neuroprotective effect of afobazole in the 6-OHDA model of Parkinson’s disease and defines the therapeutic perspective of Sigma1R agonists in the clinic.                                                                                                                                                

Sigma-1 (σ1) Receptor in Memory and Neurodegenerative Diseases

The sigma-1 (σ₁) receptor has been associated with regulation of intracellular Ca²⁺ homeostasis, several cellular signaling pathways, and inter-organelle communication, in part through its chaperone activity. In vivo, agonists of the σ₁ receptor enhance brain plasticity, with particularly well-described impact on learning and memory. Under pathological conditions, σ₁ receptor agonists can induce cytoprotective responses. These protective responses comprise various complementary pathways that appear to be differentially engaged according to pathological mechanism. Recent studies have highlighted the efficacy of drugs that act through the σ₁ receptor to mitigate symptoms associated with neurodegenerative disorders with distinct mechanisms of pathogenesis. Here, we will review genetic and pharmacological evidence of σ₁ receptor engagement in learning and memory disorders, cognitive impairment, and neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, and Huntington’s disease.

Crosstalk between endoplasmic reticulum stress and oxidative stress in schizophrenia: The dawn of new therapeutic approaches

Highlights

The complete understanding of the pathways and the point of convergence of ER and oxidative stress in schizophrenia is still quite fragmentary.

Neuronal migration along with altered secretion of neurotrophins modulates neuronal circuits and synaptic function during schizophrenia.

Chemical chaperones including Sigma-1 receptor agonists may prevent stress-induced protein misfolding associated with schizophrenia.

ER-stress inhibitors, sigma-1 receptor agonists and gene therapies holds a strong therapeutic potential against schizophrenia.

Disruption of oxidant/anti-oxidant ratio as well as endoplasmic reticulum (ER) stress are thought to be involved in the pathophysiology of schizophrenia. These stresses can lead to impairments in brain functions progressively leading to neuronal inflammation followed by neuronal cell death. Moreover, the cellular stresses are interlinked leading us to the conclusion that protein misfolding, oxidative stress and apoptosis are intricately intertwined events requiring further research into their mechanistic and physiological pathways. These pathways can be targeted by using different therapeutic interventions like anti-oxidants, sigma-1 receptor agonists and gene therapy to treat the neurodegenerative course of schizophrenia. We have also put empahsis on use of synthetic and natural ER stress inhibitors like 4-phenylbutyrate or salubrinal for the treatment of this disorder. This would provide an opportunity to create new therapeutic benchmarks in the field of neuropsychiatric disorders like schizophrenia, dissociative identity disorder and obsessive compulsive disorder.

                                                                                      

Targeting ligand-operated chaperone sigma-1 receptors in the treatment of neuropsychiatric disorders

Current conventional therapeutic drugs for the treatment of psychiatric or neurodegenerative disorders have certain limitations of use. Psychotherapeutic drugs such as typical and atypical antipsychotics, tricyclic antidepressants, and selective monoamine reuptake inhibitors, aim to normalize the hyper- or hypo-neurotransmission of monoaminergic systems. Despite their great contribution to the outcomes of psychiatric patients, these agents often exert severe side effects and require chronic treatments to promote amelioration of symptoms. Furthermore, drugs available for the treatment of neurodegenerative disorders are severely limited.

Areas covered

This review discusses recent evidence that has shed light on sigma-1 receptor ligands, which may serve as a new class of antidepressants or neuroprotective agents. Sigma-1 receptors are novel ligand-operated molecular chaperones regulating a variety of signal transduction, ER stress, cellular redox, cellular survival, and synaptogenesis. Selective sigma-1 receptor ligands exert rapid antidepressant-like, anxiolytic, antinociceptive and robust neuroprotective actions in preclinical studies. The review also looks at recent studies which suggest that reactive oxygen species might play a crucial role as signal integrators at the downstream of Sig-1Rs

Expert opinion

The significant advances in sigma receptor research in the last decade have begun to elucidate the intracellular signal cascades upstream and downstream of sigma-1 receptors. The novel ligand-operated properties of the sigma-1 receptor chaperone may enable a variety of interventions by which stress-related cellular systems are pharmacologically controlled.

Sigma-1 receptor ligands

Sigma-1 receptor ligands used in recent preclinical studies (2007~2010)

Clinically used drugs:

·         Afobazole (5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole dihydrochloride): Anxiolytic drug

·         Carbetapentane: Cough suppressant

·         Dextromethorphan (DM): Antitussive drug; DM-quinidine (Q) therapy is effective in reducing pseudobulbar affect in ALS and multiple sclerosis

·         Donepezil: Sigma-1 agonist; acetylcholine esterase inhibitor used in Alzheimer’s disease

·         Fluvoxamine: Clinically used SSRI; Sig-1R agonist

·         Sertraline: Clinically used SSRI with a putative Sig-1R antagonist property

·         Haloperidol: Clinically used antipsychotic; potent, but non selective sigma antagonist

·         Haloperidol-metabolite II (reduced HP, 4-(4-chlorophenyl)-alpha-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol): In contrast to haloperidol, having higher selectivity to Sig-1Rs

·         Memantine: A novel Alzheimer’s disease medication blocking NMDA glutamate receptors

·         Zonisamide: Anti-Parkinson drug approved in Japan

Involvement of endoplasmic reticulum stress and neurite outgrowth in the model mice of autism spectrum disorder

Implication of Endoplasmic Reticulum Stress in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is categorized as a neurodevelopmental disorder according to the Diagnostic and Statistical Manual of Disorders, Fifth Edition and is defined as a congenital impairment of the central nervous system. ASD may be caused by a chromosomal abnormality or gene mutation. However, these etiologies are insufficient to account for the pathogenesis of ASD. Therefore, we propose that the etiology and pathogenesis of ASD are related to the stress of the endoplasmic reticulum (ER). ER stress, induced by valproic acid, increased in ASD mouse model, characterized by an unfolded protein response that is activated by this stress. The inhibition of neurite outgrowth and expression of synaptic factors are observed in ASD. Similarly, ER stress suppresses the neurite outgrowth and expression of synaptic factors. Additionally, hyperplasia of the brain is observed in patients with ASD. ER stress also enhances neuronal differentiation. Synaptic factors, such as cell adhesion molecule and shank, play important roles in the formation of neural circuits. Thus, ER stress is associated with the abnormalities of neuronal differentiation, neurite outgrowth, and synaptic protein expression. ER stress elevates the expression of the ubiquitin-protein ligase HRD1 for the degradation of unfolded proteins. HRD1 expression significantly increased in the middle frontal cortex in the postmortem of patients with ASD. Moreover, HRD1 silencing improved the abnormalities induced by ER stress. Because other ubiquitin ligases are related with neurite outgrowth, ER stress may be related to the pathogenesis of neuronal developmental diseases via abnormalities of neuronal differentiation or maturation.

Sigma-1 receptor: The novel intracellular target of neuropsychotherapeutic drugs

The sigma-1 receptor localized at the ER modulates via its chaperone activity inter-organelle communications. Sigma-1 receptors thus regulate a variety of cellular events, such as neuronal differentiation, cellular survival, and bioenergetics. By numerous animal studies, these actions of the sigma-1 receptors have been linked to the pathophysiology of certain human diseases such as depression, ischemia, drug abuse, pain, and cancer. Considering the current pharmacotherapy of neuropsychiatric diseases that largely depends on drugs developed based on the monoamine theory, the sigma-1 receptor is expected to serve as a molecule, which provides a novel target of “post-monoamine” drugs, thus bringing a new approach for treatment of patients suffering from neuropsychiatric diseases.

                                                                                                                       

Fig. 1. Molecular functions of the sigma-1 receptor. The sigma-1 receptor possesses two transmembrane domains and mainly localize at the ER membrane. Sigma-1 receptors are clustered at the mitochondria-associated ER membrane (MAM) and ER membranes juxtaposing postsynaptic density of specific types of neurons. The ER lumenal domain of the sigma-1 receptor exerts chaperone activities by which ER membrane proteins are stabilized. The figure depicts the recently reported actions of the sigma-1 receptors including: 1) Sigma-1 receptors associating with BiP stabilizes IP₃ receptors type-3 (IP3R) at the MAM, leading to regulation of Ca²⁺ influx into mitochondria and following ATP production; 2) Sigma-1 receptors stabilize the ER stress sensor IRE1 at the MAM in an ROS-dependent manner, leading to prolongation of the IRE1-XBP1 cell survival signal; 3) Sigma-1 receptors suppress generation of reactive oxygen species (ROS) and following activation of the NFkB signaling (How the sigma-1 receptor regulates ROS generation is unknown); 4) Sterols such as 25-hydroxycholesterol promote the association of sigma-1 receptors with Insig-1 [Collaborating with Insig-1, sigma-1 receptors regulate ER-associated degradation (ERAD) of HMG-CoA reductase and galactosylceramide synthase at the ER]; 5) Sigma-1 receptors regulate the trafficking of potassium channel subunits from the ER to the plasma membrane or processing/secretion of brain-derived trophic factor (BDNF). Sigma-1 receptors likely associate with potassium channel subunits or pro-BDNF at the ER. In spinal neurons, sigma-1 receptors, which colocalize with a K channel subunit are clustered at the ER membrane apposing postsynaptic densities (PSD). How the sigma-1 receptor regulates processing/secretion of BDNF is unknown.  (in the earlier part of this post the mechanism that increases BDNF is explained, if you activate sigma-1R you inevitably will increase BDNF)

Conclusion

In our simplified view of autism, aimed at actually treating it, we should have a list of stresses and what to do about them:-

·        Oxidative stress

·        Nitrosative stress

·        Endoplasmic Reticulum (ER) stress

Reducing oxidative stress has multiple biological and behavioral effects; the overall effect is generally positive.

Reducing endoplasmic reticulum (ER) stress, if present, does look a good idea.  It will have numerous effects; it should even reduce oxidative stress. The sigma-1 chaperone looks like it will have many effects that, on balance, should be positive, but undoubtedly may upset something and produce an overall negative effect in some people – it is inevitable.  I hope the effect on NMDA receptors does not cause a problem where an E/I (excitatory/inhibitory) imbalance is already being treated.

A highly selective sigma-1R agonist, one that does not affect any other receptors, does not exist.

Many psychiatric drugs like antidepressants do affect sigma-1R, but they are not suitable for long term use because of side effects, tolerance, addiction etc.

Afobazole is interesting because clinical trials have shown it to be well tolerated, non-addictive and reasonably effective for the treatment of anxiety.  Afobazole also affects the melatonin receptor MT1, it is not directly sedating but might affect some types of sleep abnormality. 

Afobazole is only researched in Russia, but findings are shared internationally, for example at this conference

The drug was developed, and is currently researched, by the “Research Zakusov Institute of Pharmacology” in Moscow. They recently also published a paper on the use of Afobazole in Parkinson’s disease.  In the Parkinson’s paper (https://www.nature.com/articles/s41598-019-53413-w) the researchers argue the role of the drug is in targeting ER stress, protein misfolding, IP3R etc.  The very things I am suggesting may be relevant to autism in today’s post.

Another interesting drug from the former USSR, though actually from Latvia (now in the European Union) is Mildronate.  I did suggest a long time ago, based on the research studies, that this might be effective to treat people with a lack of the Mitochondrial Complex 1.

I think mitochondrial disease is likely over diagnosed by MAPS-type doctors, but it is a genuine cause/contributing factor to some people’s autism.

So many people are using Mildronate to boost sporting performance and some for academic performance, it is now widely available from the same vendors /platforms selling Afobazole. (eBay, Amazon etc)

The underlying message is that when considering repurposing safe old drugs to treat neurological conditions, consider all of them, including Japanese, Russian and indeed Latvian.

Many interesting novel substances are mentioned in this blog, like Basmisanil  a highly selective negative allosteric modulator of α₅ subunit-containing GABA_A receptors for the treatment of cognitive impairment specifically associated with Down syndrome.  The problem with such novel substances is that they will be ultra expensive and often they fail in their clinical trials and are never commercialized.  Roche cancelled Basmisanil because it failed in the Down Syndrome trial.  Tuning down the response from GABAa receptors containing the α₅ subunit may very well be an effective way to improve cognitive function in some people, but the failure of this trial likely means no new substances will be developed.

While it is okay to write about new drugs in development, the real interest is in applying what can be used today. All four of the following need to be satisfied:

1.     Safe (no/minimal side effects, no tolerance, no addiction, interactions)

2.     Affordable

3.     Available

4.     Effective

Some drugs, not commonly used in Western countries, likely do tick the first 3 points, whether effective in autism depends on the individual sub-type.  Many do look interesting - from Ibudilast (Japan), to Mildronate (Latvia) for Complex 1 mitochondrial disease and perhaps Afobazole (Russia) for some schizophrenia/autism.

Afobazole is a cheap over-the-counter anxiety treatment in Russia.  It is apparently “effective” in the BALB/c mouse model, that may be relevant to autism. BALB/c mice show low sociability, relatively high levels of anxiety and aggressive behaviors, large brain size, underdevelopment of the corpus callosum, and low levels of brain serotonin.

Is Afobazole the answer to ER stress in autism?  If not, then what might be?  The schizophrenia research suggests 4-phenylbutyrate, salubrinal, cordycepin, taurosodeoxycholic acid.  Cordycepin comes from a mushroom that I recall one of our Aspie readers favours.   

This post could go on forever; I think I have made my point, but a little more:-

The lipophilic 4-phenylbutyric acid derivative prevents aggregation and retention of misfolded proteins 

Chemical chaperones prevent protein aggregation. However, the use of chemical chaperones as drugs against diseases due to protein aggregation is limited by the very high active concentrations (mM range) required for mediating their effect. One of the most common chemical chaperones is 4-phenylbutyric acid (4-PBA). Despite its non-favorable pharmacokinetic properties, 4-PBA was approved as a drug to treat ornithine cycle diseases. Here we report that 2-isopropyl-4-phenylbutanoic acid (compound 5) was (2-10 fold) more effective than 4- PBA in several in vitro models of protein aggregation. Importantly, compound 5 reduced the secretion rate of autism-linked Arg451Cys Neuroligin3 (R451C NLGN3).


Protein misfolding, detectable in blood samples, predicts Alzheimer's Disease up to 14 years before onset, perhaps in time to start effective therapy? perhaps targeting sigma-1R, or perhaps with betanin, that pigment in beetroot, that seems to disrupt plaque formation.

Protein misfolding as a risk marker for Alzheimer's disease

                                                           

In symptom-free individuals, the detection of misfolded amyloid-beta protein in the blood indicated a considerably higher risk of Alzheimer's disease -- up to 14 years before a clinical diagnosis was made. Amyloid-beta folding proved to be superior to other risk markers evaluated.