Alzheimer's Afternoons Seminar Series Summaries

Insights and discussion from the cutting edge with reference to journal articles and other research papers.
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Alzheimer's Afternoons Seminar Series Summaries

Postby Fiver » Fri Apr 17, 2020 9:52 am

I wrote up my notes from these recent online seminars, for friends and colleagues (many of whom who are not experts in the field). Pasted here in case anyone is interested.

New developments in our understanding of ApoE and late onset Alzheimer’s disease

In early 2020 a special issue of the journal Neurobiology of Disease was published containing eleven articles exploring the role of the ApoE risk gene in the development of late onset Alzheimer’s disease. This open access journal issue was edited by Allan Butterfield and Lance Johnson and can be found here:

https://www.sciencedirect.com/journal/n ... 100&page=1

This was followed by the establishment of a popular online seminar series, Alzheimer's Afternoons, organized by Lance Johnson and Rik van der Kant. This biweekly seminar series began in March – in the middle of the COVID-19 outbreak, when most researchers had closed their laboratories – and is scheduled to run into June, at least. It includes seminars by over thirty active researchers in the field, broadcast from their homes as they shelter in place, and has attracted well over 200 attendees per seminar. The series also grew to include a separate collection of shorter “happy hour” seminars presented online on Fridays by researchers-in-training. Recordings of these seminars are available online, for a limited time.

Alzheimer's Afternoons 3:00pm ET Tuesdays and Thursdays

On Twitter @AlzAfternoons

http://www.ljohnsonlab.com/ad-seminar-series.html


This confluence of articles and seminars highlights the increasing amount of research aimed at understanding the role of the apoe4 risk gene in the development of Alzheimer’s disease and related pathologies.

The published studies, useful reviews, and online seminars are all fascinating. However, it’s easy to become overwhelmed by the amount of information – two forty-minute seminars plus two shorter “happy hour” talks from trainees each week, plus the flurry of new publications. To help, I’ve produced the following summaries to help keep them organized in my mind as well as to assist those who are not experts in the field but have a personal interest in apoe4 to follow along.

For non-experts in the field:

For those starting out and looking for a good review of the apoe4 gene as related to Alzheimer’s disease I recommend this recent review:

A Quarter Century of APOE and Alzheimer’s Disease: Progress to Date and the Path Forward
Michael E. Belloy, Valerio Napolioni, and Michael D. Greicius
Neuron 101, March 6, 2019
https://doi.org/10.1016/j.neuron.2019.01.056

For those who are not scientists in closely related fields – or those who are healthcare providers, policy-makers, advocates, caregivers, apoe4 carriers, or concerned family members – I offer the following recommendations:

• Most articles discussed here are “open access”, meaning free to download and read. A few might be behind a paywall. For those articles behind a paywall you can access a summary and sometimes the figures. Also, you will find that most researchers are willing top provide free PDF copies of their “paywalled” papers if you ask nicely. (Of course, it’s an interesting question why any taxpayer funded research would be locked up behind a paywall in the first place, but that’s a subject for another time.)

• This research is quite difficult. It requires adequate funding and long periods of time to build and maintain a functioning laboratory of graduate students, technicians, and post-doctoral researchers. These individuals train for decades, work very long hours, and endure far more failure than success. The articles you read are triumphs of tenacity and collaboration. The best way to ensure that their progress continues is to support basic science research. This is especially important now, when many researchers can not access their laboratories due to the COVID-19 pandemic.

• Don’t be overwhelmed by the complexity of research articles, thinking that you (or anyone else) should understand them upon a first reading. We’re here to help! I remind undergraduate science students that they are doing well to understand half of what they read the first time through, and 75% upon more careful study. Very few people – maybe the authors, if not too much time has passed – will understand >90% of any published article. It’s ok to rely upon the summaries, abstracts, and good science news coverage of an article.

• The process of translating basic research into medical care is a long and arduous one. Clinical trials take many years and millions (sometimes billions) of US dollars and many years to complete. Most potential therapies fail – this is something all too familiar to anyone following Alzheimer’s research. But such trials are necessary to turn basic research into an effective and safe treatment. The research described here represent important first steps on this journey.

• The usual disclaimer applies: Nothing here should be taken as medical advice.

• If you wish to support Alzheimer’s research by volunteering for a clinical trial, here’s one way to find more information: https://www.actcinfo.org/.

• If you are searching for a supportive community of people who carry the apoe4 risk gene, try: http://www.apoe4.info.
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Re: Alzheimer's Afternoons Seminar Series Summaries

Postby Fiver » Fri Apr 17, 2020 9:55 am

Alzheimer’s Disease: Tipping the energy balance
Alzheimer’s Afternoons Seminar Series (March 26)

The Alzheimer’s Afternoons Seminar Series started off with a seminar by Dr. Lance Johnson from the University of Kentucky. Dr. Johnson is one of the organizers of the popular online seminar series but also a guest editor (along with Allan Butterfield) for a special issue of the journal Neurobiology of Disease containing eleven articles on ApoE and Alzheimer’s. The open access articles (technically published in June 2020, but available online in April 2020) can be accessed here:

https://www.sciencedirect.com/journal/neurobiology-of-disease/collection/10C0D9JSJQP?page-size=100&page=1

This seminar is based in part of a recent article in this series:

Holden C. Williams, Brandon C. Farmer, Margaret A. Piron, Adeline E. Walsh, Ronald C. Bruntz, Matthew S. Gentry, Ramon C. Sun, Lance A. Johnson. APOE alters glucose flux through central carbon pathways in astrocytes. Neurobiology of Disease, Volume 136, 2020, 104742, ISSN 0969-9961. https://doi.org/10.1016/j.nbd.2020.1047 ... 6120300176)

Dr. Johnson opened by explaining the theory that carriers of the apoe4 risk gene, which significantly increases the incidence of late onset Alzheimer’s disease (LOAD), process metabolic "fuels" differently. Specifically, he cited increasing evidence that apoe4 astrocytes and microglia – brain cells which tend to neurons and fight infection - are less able to process sugars by the usual processes of glycoloysis, the citric acid cycle, and/or oxidative phosphorylation (OXPHOS). He postulated that this one reason why apoe4 astrocytes fail to adequately nurture and protect neurons.

He mentioned the Randle cycle (the “glucose fatty-acid cycle”) which helps cells fine-tune fuel usage. Normally, this cycle balances the competition of glucose and fatty acids for metabolic substrates in cells. (If you own a plug-in hybrid car think of this as the computer which constantly balances the use of the engine vs. the battery to drive the wheels.)

In recent years the inability to use glucose effectively has become a well-recognized hallmark of the disease.

Dr. Johnson hypothesized that apoe4 brain cells process glucose mainly through glycolysis, ending in the production of lactate.
Normally, this occurs when adequate oxygen for OXPHOS is lacking. It can also occur in cancerous tumors via the Warburg effect. In any case, converting glucose to lactate provides only a small amount of energy and requires that lactate – which becomes toxic when it accumulates - be recycled later, at significant metabolic cost. In short, it is a quick way to produce a bit of ATP and NAD, but quite inefficient.

His theory also predicts that apoe4 brains would use fats as an alternative fuel.

This may require some explanation for non-experts:

Fatty acids are simple chains of hydrocarbon molecules, with a small “head group”. They vary in length from short chain (e.g., having eight carbons; C-8) to long chain (e.g., having 18 carbons; C-18) and usually have an even number of carbons in the chain. Sometimes specific carbons are connected by double bounds, instead of single bonds; these “degrees of unsaturation” form kinks in the chain and change the molecule’s personality.

Double bond kinks can determine how fats “stack” or fit together. Fatty acids that stack well tend to be solid at room temperature and are called “fats”, whereas those with double bond kinks tend to require more personal space, are liquids at room temperature, and are commonly called “oils”.

Examples include: saturated fats (no double bonds), unsaturated fats (one double bond), poly-unsaturated fats (multiple double bonds), and trans- and partially hydrogenated- fats (mostly commercial food products; not healthy!), as well as omega-3 fatty acids (often anti-inflammatory) and omega-6 fatty acids (often pro-inflammatory).

For convenient storage fatty acids can be “hung” from a glycerol molecule – like belts on a clothes hanger. These are the monoglycerides (one hanging chain), diglycerides (two hanging chains), triglycerides (three hanging chains, good for storage and transport), or phospholipids (two hanging chains and a phosphate head group).

“Lipid” is a very general term. Lipids are, technically, anything similar that doesn’t dissolve easily in water – such as fatty acids, waxes, sterols, fat-soluble vitamins (A, D, E, and K), monoglycerides, diglycerides, triglycerides, and phospholipids.
By the way cholesterol is not a fat. It has a basic steroid structure, and is synthesized differently.

Brain tissues usually prefer to burn sugars as fuel. Burning fats as fuel first requires that larger triglycerides and longer chain fatty acids be trimmed to a size that can enter the mitochondria. Then is requires beta-oxidation of shorter-chain fatty acids inside mitochondrial matrix, a process that is slower and can produce more toxic reactive oxygen species (ROS) than the burning of sugars.

You’ll recall that fatty acids can be obtained from the diet and transported in the blood as triglycerides. Triglycerides are “oily” and not soluble in blood plasma; hence they are circulated (along with cholesterol) inside tiny lipoprotein particles. These particles include the familiar LDL (the “bad” cholesterol carrier) and HDL (the “good” cholesterol carrier), and many others. The liver is one of the main sources of cholesterol and lipids and acts as a distribution hub to build, export, and recycle apolipoprotein particles.
These particles contain some proteins – these serve as a scaffolding to help shape the particles into hollow spheres and also acts as an address label of sorts. Cells hoping to import cargo carried in lipoprotein particles display receptors to these proteins on their surfaces – and snag the particles as they pass by. ApoE (apolipoprotein E) is one of these proteins, present on the surface of many (but not all) types of lipoprotein particles. The apoe4 version of this protein differs only by one or two amino acids, but this give the molecule a different shape and different binding tendencies.

These familiar types of lipoprotein particles produced primarily by the liver - LDL, VLDL, IDL, and HDL - do not usually penetrate the blood-brain barrier. Thus, the brain is “cut-off” from dietary fats and lipids. Instead the brain produces its own fats, lipids, and cholesterol and shares them among the various types of brain cells in analogous lipoprotein particles that occur only in the brain. However, the apoe4 protein is a key component of lipoprotein particles found both inside and outside of the brain.
In some cases, brain cells that are particularly hungry for fats to burn as fuel can end up breaking down the protective myelin sheaths of neurons into free fatty acids. This would be problematic, of course.

Dr. Johnson noted the Dr. Alois Alzheimer observed "lipid droplets" in the brain of patients in 1907. Dr. Johnson’s research group wondered if these “droplets” were accumulations of fats and they wondered if individuals carrying apoe4 genes, and having a higher risk of developing LOAD, accumulated more lipid droplets in brain tissues. By analyzing brain tissues, they found interesting differences: apoe4 brain cells accumulation more droplets, but they are also smaller in diameter compared to apoe3 brain cells.

This might be explained in part by observation that apoe4 cells have lower activity of ATP-binding cassette transporters (ABC transporters) which facilitate lipid efflux from cells. ABC transporters load fats and lipids into lipoprotein particles for export; the presence of apoe4 reduces this, leaving apolipoproteins underinflated (or “poorly lipidated”, to use the technical term). This could explain the group’s observation.

It would be expected that lipid droplets would accumulate inside apoe4 cells if lipids and fatty acids could not be exported as rapidly.

Dr. Johnson’s group noted that the accumulation of small lipid droplets in apoe4s worsen with age.

In another set of experiments, they fed apoe4 brain cells fatty acids labeled with radioactive carbon (14C) - a useful way to trace the path of these compounds in cells. They found that apoe4 cells were indeed burning more fatty acids by beta-oxidation in mitochondria, supporting their hypothesis.

To explore this further they conducted metabolic experiments on 94 human subjects (all <55 years old and healthy; 33 of these carried at least one copy of the apoe4 gene). The researchers recorded the subject’s metabolic activity – including their respiratory exchange ratio - at rest, then after consuming sugary drinks. They found that subjects without apoe4 genes (including those with the apoe2 genes, which is protective against LOAD) burned the sugars easily. They showed the typical increase in oxygen uptake that is expected when subjects are burning sugars as fuels. On the other hand, subjects with apoe4 genes did not metabolize sugars as well; they exhibited no increase in oxygen uptake or usage.

Their conclusion from this collection of experiments - some of which were recently published - was that the data support the original theory: apoe4 cells process sugars poorly and seem to use more fatty acids as fuel to compensate.

Dr. Johnson compared the metabolism of apoe4 astrocyctes and microglia to the "Warburg effect" seen in cancerous tumors. As mentioned previously many types of cancerous cells run glycolysis without the citric acid cycle and OXPHOS. This process is fast but inefficient. It churns out lactate acid as a waste product. He mentioned that a “high fat diet” made this more pronounced. NOTE: a “high-fat diet” usually refers to a poor-quality diet akin to a “Super-size Me”-type Standard American Diet, which is appropriately abbreviated as S.A.D.
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Re: Alzheimer's Afternoons Seminar Series Summaries

Postby Fiver » Fri Apr 17, 2020 9:56 am

Alzheimer’s Disease: next generation mouse models
Alzheimer’s Afternoons Seminar Series (March 31)

This seminar was presented by Dr. Gareth Howell, Associate Professor and the Diana Davis Spencer Foundation Chair for Glaucoma Research at the Jackson Lab. His premise was that progress in AD research has been hindered by poor mouse models. He is a member of the MODEL-AD group, a collection of hundreds of researchers working to create and test next-generation mouse models.

He began by describing new mouse models developed with human apoe4 genes.

Murine apoE (the gene in mice) and human apoE4 are not the same, making wild type mice relatively useless for the study of Alzheimer’s disease. For example, murine apoe4 vs human apoe4 have quite different tendencies to facilitate plaque or CAA formation. As result, transgenic mice wherein murine apoe genes are replaced by human apoe4 (or apoe3 or apoe2) genes are very valuable.

Thanks to the work of those at the Jackson Labs (and many other institutions) many other types of mice expressing other AD risk genes are available.

He noted that mice carrying human apoe4 genes exhibit a break-down in brain vessels, specifically fibrin leakage into the brain, but only in mid-life mice (not at younger ages). In these mice blood flow was reduced, as visualized by PET scans. They are currently exploring the potential benefits of exercise to correct this in 4/4 (vs. 3/3) mice. Thus far, they have found that apoe4/4 mice are just as good at exercise and had no observed differences when young. They also found that, in these mice, exercise helped to prevent the problems apoe4/4 mice normally developed with increasing age but only if the exercise was started when mice were young.
In general, he noted that neither human apoe4/4 genes nor older age are sufficient by themselves to cause AD in these mice. Some other trigger is necessary.

They also developed mice carrying human TREM2 genes.

TREM2 is shorthand for “triggering receptor expressed on myeloid cells 2”. It is a receptor found on the surface of many types of cells, including microglia, that is important for the functioning of the innate immune system. Some alleles (genetic variants) of the TREM2 gene are associated with late-onset Alzheimer’s disease and related dementias.

Dr. Howell made crosses to create of mouse populations with particular combinations of APOE and TREM2 genotypes. They measured mouse "frailty" in these various populations and noted no differences. They also noted that apoe4 protein levels – the total amount of apoe protein measured – don't seem to change with age in these mice.

Dr. Howell observed that amyloid beta accumulation seems to be a trigger to cause AD symptoms - but this is often inconsistent. This is in line with many previous observations that the accumulation of Aβ in the brain is often, but not always, associated with cognitive decline. Aβ accumulation is just one possible – though probably common – trigger.

Together, this large group of researchers collected data suggesting that apoe4 mice tend to have a more fragile central nervous system, especially when older.

The most impressive aspect of Dr. Howell’s seminar was the enormous amount of work done with the MODEL-AD group to generate better mouse models of LOAD.
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Re: Alzheimer's Afternoons Seminar Series Summaries

Postby Fiver » Fri Apr 17, 2020 9:59 am

Cholesterol, Apoe4, and Alzheimer’s disease
Alzheimer’s Afternoons Seminar Series (April 2)

This seminar was presented by Dr. Rik van der Kant, a Research Associate in both the Faculty of Science, (Functional Genomics) and Amsterdam Neuroscience (Neurodegeneration) at Vrije Universiteit Amsterdam. He is an expert on cholesterol metabolism and is studying how apoe4 contributes to LOAD. The title of his seminar was “Cholesterol metabolism as a dual driver of neuronal AD pathology”. He described his recent work that was published (open access) here:

van der Kant, R.; Langness, V. F.; Herrera, C. M.; Williams, D. A.; Fong, L. K.; Leestemaker, Y.; Steenvoorden, E.; Rynearson, K. D.; Brouwers, J. F.; Helms, J. B.; Ovaa, H.; Giera, M.; Wagner, S. L.; Bang, A. G.; Goldstein, L. S. B. Cholesterol Metabolism Is a Druggable Axis That Independently Regulates Tau and Amyloid-β in IPSC-Derived Alzheimer’s Disease Neurons. Cell Stem Cell 2019, 24 (3), 363-375.e9. https://doi.org/10.1016/j.stem.2018.12.013.

Dr. van der Kant began by reviewing how human cells can be converted in stem cells, then into almost any other type of cells. This is useful for making populations of neurons, astrocyctes, and microglia carrying apoe4 genes. This work is described in another recent article, found here:

van der Kant, R.; Goldstein, L. S. B.; Ossenkoppele, R. Amyloid-β-Independent Regulators of Tau Pathology in Alzheimer Disease. Nat. Rev. Neurosci. 2020, 21 (1), 21–35. https://doi.org/10.1038/s41583-019-0240-3.

He explained that when such cells are created from patients with LOAD they produce high levels of amyloid beta (Aβ40) and hyperphosphorylated tau (p-tau) - hallmarks of the disease.

Recall that amyloid beta is a peptide fragment of the large amyloid precursor protein (APP), which occurs on many types of cells. APP is cleaved by the enzymes beta secretase and gamma secretase to yield Aβ. Amyloid beta fragments can be 36 to 43 amino acids long, with Aβ40 and Aβ42 the mostly commonly studied forms. These fragments can exist as a single monomer unit, or combine to form larger oligomers, fibrils and plaques. Misfolded oligomer fragments can act as “seeds” inducing other amyloid beta fragments to misfold and aggregate, a process akin to a prionic infection.

(This is easily imagined by recalling the Slinky, a once-popular children’s toy – essentially a coil of wire which “walked down stairs, alone or in pairs” until becoming a tangled mess. Imagine placing a tangled Slinky (a “seed”) into the drawer of new, perfectly-coiled Slinkys. The inevitable outcome is a drawer full of “misfolded and aggregated” toys.)

Tau proteins are essential components of cell cytoskeletons, specifically the assembly of microtubules. However, tau proteins that have become hyperphosphorylated – covered in extra phosphate groups – also tend to misfold and stick together, becoming insoluble aggregates called neurofibrillary tangles.

Dr. van der Kant’s group used these populations of human cells grown in culture to test 1684 potential drugs to determine if they could lower either Aβ40 or p-tau. They identified 160 promising compounds, including 42 of which might be safe and/or effective in patients.

Interestingly, all statin drugs they tested were effective. This is explained more fully in their article published in the journal Cell Stem Cell. He mentioned that in previous studies statins have been shown to reduce AD risk. In their cell cultures, several statins consistently reduced p-tau accumulation in a dosage dependent manner. He noted that atorvastatin stood out as being just a bit better than the others in this regard.

This has also been noted in previous studies, including:

“Results of this analysis revealed that statin users had better cognitive scores than nonusers; and this effect was somewhat more evident with the use of some lipophilic statins (atorvastatin and lovastatin).” Geifman, N., Brinton, R.D., Kennedy, R.E. et al. Evidence for benefit of statins to modify cognitive decline and risk in Alzheimer’s disease. Alz Res Therapy 9, 10 (2017). https://doi.org/10.1186/s13195-017-0237-y

However, other studies reporting that statins reduced the risk of developing LOAD found no clear differences between the various hydrophilic and lipophilic statins; for example:

Haag, M. D. M.; Hofman, A.; Koudstaal, P. J.; Stricker, B. H. C.; Breteler, M. M. B. Statins Are Associated with a Reduced Risk of Alzheimer Disease Regardless of Lipophilicity. The Rotterdam Study. Journal of Neurology, Neurosurgery & Psychiatry 2009, 80 (1), 13–17. https://doi.org/10.1136/jnnp.2008.150433.

Probing the system further Dr. van der Kant’s group found that cholesterol itself was not the critical link to Aβ and p-tau accumulation in the brain. Rather they found that levels of cholesterol esters (CEs), a storage and transport form of the molecule, was key.

Cholesterol is an essential building block of animal cell membranes, and can be obtained from the diet or synthesized by cells. It is required for the synthesis of steroid hormones and vitamin D. It is not a water-soluble molecule and it is transported in blood within bubble-like lipoprotein particles. The brain is a cholesterol hog. It possesses 20% of the bodies cholesterol, by weight.
When cholesterol is bound to a fatty acid chain by an ester bond, cholesterol esters are formed. Most cholesterol in our diets and in circulating lipoprotein particles are in this form.

These researchers found that lowering CE concentrations using statins - again in cells cultures from AD patients - lowers Aβ and p-tau levels. These two effects seem to occur separately, by independent mechanisms.

Statin drugs inhibit the HMG CoA reductase enzyme, the rate limiting step in cholesterol production.

One interesting finding is that the amyloid precursor protein (APP; the cell surface protein that can be cleaved to form Aβ) has a molecular site that binds strongly to cholesterol. Dr. van der Kant pointed out that eliminating this binding site prevented statins from lowering Aβ levels but that in this case p-tau was still reduced. This is more solid evidence that statins reduce Aβ and p-tau in different ways, but at the same time.

Here Dr. van der Kant made an important point. He stressed that despite their ability to reduce LOAD incidence, to a modest degree, in large population studies, statins don't generally work to treat LOAD in individual patients. He suggested this is because statins either don't reach the brain in high enough concentrations or because statins can be toxic to astrocytes, brain cells which nurture and protect neurons. He shared data to demonstrate this occurring in cell culture.

Similar statin toxicity has been observed previously. For example:

“However, there are data indicating that statins can have toxic effects on brain neural cells….In general, what distinguishes the studies on protective effects of statins versus toxic effects are the drug concentrations used in the different studies.” Wood, W. G.; Eckert, G. P.; Igbavboa, U.; Muller, W. E. Statins and Neuroprotection: A Prescription to Move the Field Forward. Annals of the New York Academy of Sciences 2010, 1199 (1), 69–76. https://doi.org/10.1111/j.1749-6632.2009.05359.x.

A review of the positive and negative effects of statins for LOAD is:

Schultz, B. G.; Patten, D. K.; Berlau, D. J. The Role of Statins in Both Cognitive Impairment and Protection against Dementia: A Tale of Two Mechanisms. Translational neurodegeneration 2018, 7 (1), 5–11. https://doi.org/10.1186/s40035-018-0110-3.

In short, while large population studies sometimes find that long-term statin is associated with modest reduction in the incidence of LOAD, suggesting a potential preventative role, statins do not cure individual patients with Alzheimer’s disease and at high levels can be toxic to important brain cell types.

Dr. van der Kant’s group identified a different drug that seems to be effective, without harming astrocyctes.

Efavirnez, sold under the brand name Sustiva (among others), is an antiretroviral medication used to treat and prevent HIV/AIDS. The researchers found that it crosses the blood brain barrier and binds specifically to an enzyme found primarily in neurons to effectively reduce CE levels. This is an encouraging dataset.

He pointed out that accumulation of "lipid droplets" is a characteristic of AD first observed by Dr. Alois Alzheimer and suggested that these droplets could have been accumulations of excess CEs in the brains of LOAD patients. This observation seems to fit with the theory that reducing CE over-production could reduce the accumulation of Aβ and p-tau.

Recall that the apoe4 risk gene seems to increase lipid droplet accumulation.

For more information about lipid droplets, apoe4, and LOAD, see the summary of Dr. Lance Johnson’s seminar from March 26.
Dr. van der Kant’s group – and others – have tested Efavirnez in mice and found it effective. He suggested these previous, related studies focusing on this drug:

Mast, N.; Li, Y.; Linger, M.; Clark, M.; Wiseman, J.; Pikuleva, I. A. Pharmacologic Stimulation of Cytochrome P450 46A1 and Cerebral Cholesterol Turnover in Mice. J. Biol. Chem. 2014, 289 (6), 3529–3538. https://doi.org/10.1074/jbc.M113.532846.

Petrov, A. M.; Lam, M.; Mast, N.; Moon, J.; Li, Y.; Maxfield, E.; Pikuleva, I. A. CYP46A1 Activation by Efavirenz Leads to Behavioral Improvement without Significant Changes in Amyloid Plaque Load in the Brain of 5XFAD Mice. Neurotherapeutics 2019, 16 (3), 710–724. https://doi.org/10.1007/s13311-019-00737-0.

In concluding, Dr. van der Kant hypothesized that therapeutics such as Efavirnez, which lower CE over-production, could be most beneficial for individuals carrying one or two copies of the apoe4 risk gene. His group is hoping to continue on to clinical trials with this already-FDA approved drug. He cautions that the drug has some side effects, of course, and it remains to be seen if it is well tolerated.

NOTE: the original list of drugs with potential to lower Aβ and p-tau levels, presented by Dr. van der Kant, included some SSRIs. There are some interesting links between SSRIs, statins, and cognition – and some suggestion that they work better together to treat mood disorders. For example, see: Köhler, O.; Gasse, C.; Petersen, L.; Ingstrup, K. G.; Nierenberg, A. A.; Mors, O.; Østergaard, S. D. The Effect of Concomitant Treatment With SSRIs and Statins: A Population-Based Study. American Journal of Psychiatry 2016, 173 (8), 807–815. https://doi.org/10.1176/appi.ajp.2016.15040463.
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Re: Alzheimer's Afternoons Seminar Series Summaries

Postby Fiver » Fri Apr 17, 2020 10:02 am

Alzheimer’s disease and epilepsy: what can we learn from their similarities?
Alzheimer’s Afternoons Seminar Series (April 14)

Dr. Misha Zilberter, a staff research scientist from the Gladstone Institutes at the University of California San Francisco Medical Center, presented a seminar entitled “Brain glucose hypometabolism and network hyperexcitability in Alzheimer's disease”.

For more information consider reading their open access article:

The vicious circle of hypometabolism in neurodegenerative diseases: Ways and mechanisms of metabolic correction Yuri Zilberter Misha Zilberter 02 May 2017 https://doi.org/10.1002/jnr.24064

Dr. Zilberter began by pointing out that both Alzheimer’s disease and epilepsy are associated with neuron hyperexcitability and a reduction in glucose metabolism.

• Hyperexcitability occurs when neuron membrane potentials fall too low, causing rapid and uncontrolled nerve cell firing.
• Reduced glucose metabolism, called glucose hypometabolism, is a known characteristic of both AD and epilepsy. It occurs even at a young age in individuals carrying apoe4 genes, which significantly increase the risk of developing AD later in life.

Dr. Zilberter noted that glucose hypometabolism tends to become more pronounced with age and the degree to which this occurs seems to predict the conversion of mild cognitive impairment to AD later in life.

Here it is worth understanding how glucose metabolism normally occurs, and the speaker provided a short primer (which I have expanded a bit here):

Most tissues obtain glucose mostly from the blood. Cells normally respond to the hormone insulin – a signal that blood sugar is available – by placing GLUT4 transporters on their surfaces, allowing glucose to enter. (This is disrupted in diabetes, in various ways.) Glucose that is to be used immediately as fuel is processes by glycolysis, generating some ATP and NADH which are common energy currencies in cells. In the absence of sufficient oxygen, lactic acid is produced and the process stops here. With sufficient oxygen, however, the remnants of glucose are shuttled into the mitochondria and completely burned up in the citric acid cycle, with a lot more ATP and NADH generated by oxidative phosphorylation (OXPHOS) in the mitochondrial electron transport chain.

However, some glucose goes another way – it enters the pentose phosphate pathway where important building blocks such as nucleotides are formed and key antioxidants are produced.

If there is excess glucose, beyond what is currently needed for these processes, it can be linked together to form long chains of glycogen for storage. This occurs most often in the liver and muscles.

Conversely, if internal glucose levels fall to very low levels and the necessary glucose can not be obtained from the blood cells can starve. In response to this situation cells can work backwards - making some from raw materials by gluconeogenesis - but this requires a significant amount of energy.

As you can see, glucose hypometabolism can occur in many ways - because there is not enough sugar in the blood, because blood sugar is not properly taken up into cells, or because glycolysis, the citric acid cycle, OXPHOS, or gluconeogenesis are inhibited.
Dr. Zilberter pointed out that key steps in glucose uptake, glycolysis, and OXPHOS are inhibited in both AD and epilepsy. As a result, cells can not use glucose as efficiently, even if there is plenty around.

He noted, as an example, that children in India that eat lychees before bedtime, on an empty stomach, sometimes have seizures ending in death. This reaction is caused by the natural product hypoglycin, which inhibits the production of new glucose by gluconeogenesis during the nighttime when blood sugar can also be low (hypoglycemia). This is one example of how glucose hypometabolism can trigger epileptic seizures.

In the lab glucose hypometabolism can be simulated by the artificial compound 2-deoxy-D-glucose (2-DG), which inhibits glycolysis. The effect is mild, with glucose metabolism reduce by about 14%. Nonetheless, after 4 weeks of treatment sections of healthy brains show hyperexcited neurons and electrical seizures, similar to those found in epilepsy. This is another example of how glucose hypometabolism can cause epilepsy, or at least related symptoms.

Interestingly, simply washing healthy brain sections in amyloid beta (Aβ) has a similar effect. It induces glucose hypometabolism and hyperexcitability in nerve cells.

Dr. Zilberter noted that this occurs similarly in both epilepsy and AD. He wondered if there was a common cause that would explain the similarity.

The key observation was that just moments before seizures would occur in sectioned brain slices there was a spike of hydroperoxide (H2O2). In fact, applying H2O2 alone would trigger the response.

H2O2 is a dangerous type of reactive oxygen, which can damage cells. Tiny amounts are naturally produced in mitochondria during OXPHOS. Too much H2O2 production by old or damaged mitochondria, however, can trigger cell death, contributing to various diseases, and accelerate aging. Most healthy cells – with young, healthy mitochondria - produce as little H2O2 as possible and have various mechanisms to quickly destroy reactive oxygen species like H2O2 when they are generated. However, there are exceptions. For example, some immune cells generate H2O2 on purpose, to kill nearby microbes. (Just as we often use H2O2 to sanitize wounds or surfaces, i.e. to prevent microbial infection.) These guardian cells have NADPH oxidase (NOX) proteins on their surfaces, which generate H2O2 outside of these cells. In the brain, microglia are primary immune defense cells and possess NOX proteins to produce H2O2.

There are many types of NOX proteins; Dr. Zilberter focuses on one called NOX2 found on the surface of microglia in the brain. Here it all started to make sense. Washing healthy brain slices with Aβ causes the spike in H2O2, nerve cell hyperexcitability, and glucose hypometabolism, as expected. But when NOX2 was inhibited, and no H2O2 was produced by microglia, this was prevented. There was no nerve cell hyperexcitability, Aβ was no longer toxic, and neurons didn’t die.

The same thing could be accomplished just by reducing the number of microglia that were present.

The problem it seems is that: the brain’s microglia respond to Aβ in the brain by generating H2O2, which damaged nerve cells, causing hyperexcitability and reduced glucose metabolism. NOX2 inhibitors – such as those described here (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709343/) - might be useful in preventing this in vivo, and deserve further study. In theory, the aim would be to reduce H2O2 production by NOX2 in the brain without compromising the ability of the immune system to defend the body against infection.

For example, consider that:

Chronic granulomatous disease (CGD) is an inherited disorder of NOX2 characterized by severe life-threatening bacterial and fungal infections and by excessive inflammation, including Crohn’s-like inflammatory bowel disease (IBD). Singel KL, Segal BH. NOX2-dependent regulation of inflammation. Clin Sci (Lond). 2016;130(7):479–490. doi:10.1042/CS20150660

During a long Q&A session that followed the seminar, Dr. Zilberter made two points I found very interesting:

First, when asked if antioxidants could help prevent that accumulation of H2O2 in the brain he remarked that they aren’t, in his experience, fast enough to prevent the H2O2 spike. It is, he surmised, better to stop this from happening in the first place.

Second, he was asked about ketogenic diets, very low carbohydrate diets sometimes used to treat epilepsy and AD. He suggested that the benefit of these diets would probably be that endogenous ketones, produced by the liver and circulated in the blood, could be used as a fuel, thereby freeing up the increasingly limited glucose in apoe4 cells for use in pathways that can only be fueled by glucose.
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Re: Alzheimer's Afternoons Seminar Series Summaries

Postby Fiver » Fri Apr 17, 2020 10:04 am

Cholesterol and matrisome networks in Alzheimer’s disease
Alzheimer’s Afternoons Seminar Series (April 16)

Dr. Julia TCW is an Instructor at the Icahn School of Medicine at Mount Sinai in New York (USA). She uses human induced pluripotent stem cells (iPSC), transgenic mouse models, and human tissue samples to explore the effects of the apoe4 risk gene on gene expression and metabolism.

Her talk touches on several different projects, including some related to the following open-access articles:

Reduced variability of neural progenitor cells and improved purity of neuronal cultures using magnetic activated cell sorting. Bowles KR, Tcw J, Qian L, Jadow BM, Goate AM. PLoS One. 2019 Mar 27;14(3):e0213374. doi: 10.1371/journal.pone.0213374. eCollection 2019. Erratum in: PLoS One. 2019 Apr 25;14(4):e0216312.

Human iPSC application in Alzheimer's disease and Tau-related neurodegenerative diseases. Tcw J. Neurosci Lett. 2019 Apr 23;699:31-40. doi: 10.1016/j.neulet.2019.01.043. Epub 2019 Jan 24. Review.

Dr. TCW began with a review of microglia and astrocytes. She pointed out that both microglia and astrocytes react to nearby amyloid beta (Aβ) in the brain by changing the expression of their genes. She focused on two important processes, astrocyte clearance of Aβ and the export of cholesterol from these cells, which depend in some way or another on the apoe protein.
She reminded listeners that the apoe4 variant is the strongest genetic risk factor for late onset Alzheimer’s disease (LOAD).

She cited a very recent study on genetic risk:

Andrews, S. J.; Fulton-Howard, B.; Goate, A. Interpretation of Risk Loci from Genome-Wide Association Studies of Alzheimer’s Disease. The Lancet Neurology 2020, 19 (4), 326–335. https://doi.org/10.1016/S1474-4422(19)30435-1.

She mentioned that this study concluded that apoe4/apoe4 individuals have a 15-fold higher lifetime risk of LOAD. This is consistent with previous estimates.

After covering some of the basics of apoe4 protein structure and function, she emphasized that in the brain apoe is produced mainly by microglia and astrocyctes.

She studied the impact of 4/4 vs 3/3 genotypes in three types of models: human iPSC grown in culture, transgenic mice carrying human genes, and postmortem brain tissues from human subjects.

NOTE: it is relatively rare to find studies which focus on apoe4/apoe4 homozygotes. Most studies struggle to find enough human 4/4 carries for studies and, as a result, group 4/4s and the more common 3/4s together as “apoe4 carriers”, a shortcut bound to muddy the data. This clear distinction, in three different models, is a real strength of these studies.

When making human iPSC lines, tissue samples were obtained from human 3/3 or 4/4 subjects. Cells were converted from fibroblasts, to stem cells, to neural progenitor cells, and then to neurons and astrocytes. Microglia can be produced along the way as well. The process, which takes les than 30 days, is described in the following open-access article:

An Efficient Platform for Astrocyte Differentiation from Human Induced Pluripotent Stem Cells. Tcw J, Wang M, Pimenova AA, Bowles KR, Hartley BJ, Lacin E, Machlovi SI, Abdelaal R, Karch CM, Phatnani H, Slesinger PA, Zhang B, Goate AM, Brennand KJ. Stem Cell Reports. 2017 Aug 8;9(2):600-614. Doi: 10.1016/j.stemcr.2017.06.018. Epub 2017 Jul 27.

The group verified that the apoe4 genotype was the only genetic contributor to AD pathology in these cells. The presence of other risk genes was ruled out. Here they compared 6 vs 7 lines of cells derived from brain tissue of age-matched, cuacasian subjects with either apoe3/3 or apoe4/4 genotypes. The age of the donors was not a factor, she indicated, since iPS cells have the typical aging factors removed during the differentiation process. Finally, Dr. TCW noted that there are different types of astrocyctes and they controlled for this in their studies.

They found that:

• In apoe 4/4 microglia pathways associated with cholesterol, lipid, and steroid biosynthesis were increased while pathways associated with lysosomal recycling and cholesterol efflux were decreased.
• In apoe 4/4 astrocytes, cholesterol biosynthesis pathway genes were also increased.
• In these apoe4/4 cells several large pathway networks seemed to be altered: SREBF2 and SCAP up, POR down.
• HDL-mediated lipid transport network was inhibited in apoe4/4 microglia (mainly driven by decreased FXR/RXR activity).

As a result, we would expect cholesterol to be accumulating, perhaps to excess, in these cells which are likely to be producing it faster than they export it.

Dr. TCW displayed a diagram of CHO transport throughout cells, it illustrated the familiar process:

Cholesterol is imported by cells as LDL apolipoprotein particles bind to LDL surface receptors and are engulfed into early endosomal “bubbles” in the cytoplasm. Later, endosomal “sorting” occurs in late endosomes, before or after they combine with lysosomes. This process separates cholesterol molecules from the LDL particles, which in turn have been separated from the LDL receptors. The receptors and LDL particles can be recycled back to the cell membrane and released while CHO reaches the endoplasmic reticulum.

Excess cholesterol, beyond what can be used by the cell, forms lipid droplets (perhaps similar to those first observed by Alois Alzheimer’s in postmortem brain tissues). This cholesterol can be exported from the cell when ABCA1 proteins, located on cell membranes, load cholesterol into lipoprotein particles, which include apoe proteins on their surfaces.

Defects in various steps can cause problems. Altered binding of LDL particles to LDL receptors can impact cholesterol import. Altered sorting or disrupted release of complexes within late endosomes can create virtual traffic jams. Defects in ABCA1 and apoe proteins can inhibit cholesterol export.

Dr. TCW then introduced the concept of “matrisome pathway dysregulation” which they have observed in hiPSC mixed cultures.

As a reminder, the matrisome is the collection of proteins that are secreted from cells to form the extracellular matrix. This includes many glycoproteins and collagens. The matrisome is the subset of the larger secretome, the set of all proteins secreted into the extracellular space.

In a series of experiments the researchers examined how apoe 4/4 cells differed with regards to the matrisome and other gene networks. They used three different approaches:

1. They examined five different cell types, including microglia and astrocytes, from brains of AD patients (obtained from Mt. Sinai) having apoe 3/3 and apoe 4/4 genotypes.
2. They analyzed the matrisome in brain cells from mice – including apoe3/3, apoe4/4, and apoe knockout (KO) mice.
3. They also examined this in human cell lines with altered using CRIPSR cas9 technology.

They found that apoe4 causes lipid and cholesterol dysregulation. For instance:

Cholesterol production was often increased:
o Apoe 4/4 microglia upregulate the synthesis of cholesterol.
o Apoe4/4 astrocytes exhibit a 20% increase in free cholesterol.
o Lipoprotein cholesterol was often higher in apoe 4/4 cultures.
o The activity of HMG CoA reductase, the rate limiting step of cholesterol biosynthesis, was increased in apoe4/4 cells.
o Imaging revealed higher levels of cholesterol in apoe4/4 astrocytes.

Cholesterol export was often inhibited:
o Apoe4/4 cells often displayed decreased cholesterol efflux, levels of apoe protein, and level of ABCA1 protein.
o Apoe4/4 astrocytes displayed higher matrisome network activity, and increases in interferon and cytokines (IL-6 and IL-8) in astrocytes and/or microglia.

These numerous experiments are complicated and generated large amounts of data. For those with interest, it’s worth reading the original papers to learn more.

Taken together, the data seem to indicate that apoe4 causes changes in cholesterol metabolism, inflammation, and the construction of the extracellular matrix, to different degrees in a variety of brain cell types. Dr. TCW concluded by describing three projects which build on these observations.
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Re: Alzheimer's Afternoons Seminar Series Summaries

Postby SusanJ » Sat Apr 18, 2020 5:44 am

Fiver, many thanks for taking the time to summarize and share these seminars! Much appreciation for these perspectives.

Fiver wrote:Most studies struggle to find enough human 4/4 carries for studies and, as a result, group 4/4s and the more common 3/4s together as “apoe4 carriers”, a shortcut bound to muddy the data.


I do wish they would also stratify 3/4. Hard to know, do I have half the process/amounts/dysregulation or is it tipped in favor of one over the other? Until data are better stratified, it's also not clear to me how to manage being a "muddy" 3/4. For example, I note successes in 4/4s (like keto) that just don't seem to work as well for me.

Anyway, fascinating stuff!

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Re: Alzheimer's Afternoons Seminar Series Summaries

Postby floramaria » Sat Apr 18, 2020 3:58 pm

Fiver wrote:Here it all started to make sense.

Thank you so much for sharing the information with us, Fiver. I appreciate the time you spent summarizing these seminars. Much of it is over my head so I am grappling with understanding. Just when my eyes were beginning to roll backwards in my head, I came to
your sentence quoted above. I am happy to know that at that point , it all made sense to you! Applied to myself, that simple sentence was my favorite laugh-inducer of the week. :lol:
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Re: Alzheimer's Afternoons Seminar Series Summaries

Postby Fiver » Sat Apr 18, 2020 4:45 pm

:)

You saw that, eh?

There are about 15 more seminars coming. :shock:

I think when that's over I'll write a much shorter summary of it all.

If I had to sum it up thus far it would be:

apoe4 research is finally getting some of the spotlight and the attention it deserves.

usually some trigger is required to trigger "apoe4" problems - could be aging, stress, pollution, infection, etc. apoe4 brain are more sensitive.

apoe4 genes causes problems with cholesterol metabolism, this involves astrocytes and microglia, and it gets worse as we get older.


The seminars were very technical and the notes are mostly for my students, but I can summarize at the end.
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Re: Alzheimer's Afternoons Seminar Series Summaries

Postby BrianR » Sun Apr 19, 2020 9:24 am

Fiver wrote:I wrote up my notes from these recent online seminars, for friends and colleagues (many of whom who are not experts in the field). Pasted here in case anyone is interested.

Fiver, these notes are wonderful! Thank you so much.


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