Lactic acid, the mitotic spindle killer

Nature vol. 616 pp. 790 – 797 ’23 is one of the most interesting papers I’ve read in the past year, both for its contents and for the two very large issues it raises (which the authors don’t really discuss).

Simply stated, the rise in cellular lactic acid levels from 6  milliMolar at mitosis onset, to 15 – 20 when mitosis is nearly over is what ’causes’ the breakdown of the mitotic spindle.

It’s now 100 years since Otto Warburg noted that tumors metabolize glucose by glycolysis producing 2 molecules of ATP per glucose (and two molecules of lactic acid) when, with plenty of oxygen around, they could get 38 molecules of ATP using their mitochondria.   This is called aerobic glycolysis.

Tumors are said to be energy hungry, so why do they use aerobic glycolysis? Simply because using oxygen to chew up glucose gives you lots of ATP along with CO2 and water, leaving you nothing to build new tumor cells with.  All 6 carbons remain present after glycolysis

The last stage of mitosis is called anaphase, where the mitotic spindle (made of microtubules) is broken down, among other things such as reformation of the nuclear membrane, and separation of the two daughter cells.

Well protein breakdown immediately brings ubiquitin to mind which, when added to most proteins, targets them to the proteasome, a huge molecular complex which breaks proteins down completely to their constituent amino acids.

APC/C is another huge multiprotein complex (at least 13 different protein subunits with a molecular mass of 1.2 megaDaltons) which acts to add ubiquitin to components of the mitotic  spindle (made mostly of microtubules).  So APC/C is a ubiquitin ligase, a dangerous thing to have around most of the time, which it is why it is usually inhibited so the cell doesn’t destroy itself.

One APC/C subunit is APC4, which has ubiquitinLike molecules (SUMO) attached to two of its lysines (#722 and #798) to activate the ubiquitin ligase activity of APC/C.    APC4 is held in check by yet another enzyme, SENP1, which removes the SUMOs.

Where does lactic acid fit in to all this?  It binds to the active site of SENP1 when coordinated with zinc ions, inhibiting SENP1’s ability to remove SUMO.

Byzantine enough for you?  Lactic acid inhibits SENP1 which inhibits APC4 allowing uninhibited APC4 to activate APC/C which breaks down the mitotic spindle.

Lactic acid, if thought of at all, was regarded as an important part of cellular metabolism, not an enzyme inhibitor.   This is an example of moonlighting, a lot of which goes on in the cell. https://luysii.wordpress.com/2021/05/04/is-there-anything-in-the-cell-that-has-just-one-function-more-moonlighting-this-time-mrna/  with its links will get you started.

Here is one of the larger issues the paper raises — how events in the cell at all levels of structure are linked to each other.  Phillip Anderson famously said “More is Different”.  The paper shows how something very small (lactic acid fits into a 5 Angstrom (.5 nanoMeters) sphere) and yet  is responsible for breaking down something 40,000 – 100,000 times larger  (the length of a microtubule in the mitotic spindle).

Here is the other (even larger) issue — Lactic acid was found as a player in cell metabolism, e.g., it is a member of the metabolome.  I was amazed to find out how large it is — some 42,000 for in the Human Metabolome DataBase http://www.hmdb.ca/metabolites?c=hmdb_id&d=up&page=1676 — for details please see https://luysii.wordpress.com/2015/06/03/how-little-we-know-2/.  Not only do we not know what they are doing, we don’t even know the structure of most of them. State of the art untargeted metabolomics studies still report ‘up to’ 40% unidentified, but potentially important metabolitcs which can be detected reproducibly. The unknown metabolites are only rarely characterized because of the extensive work required for de novo structure determination..

Do glia think? Take II

Do glia think Dr. Gonatas?  This was part of an exchange between G. Milton Shy, head of neurology at Penn, and Nick Gonatas a brilliant neuropathologist who worked with Shy as the two of them described new disease after new disease in the 60s ( myotubular (centronuclear) myopathy, nemaline myopathy, mitochondrial myopathy and oculopharyngeal muscular dystrophy).

Gonatas was claiming that a small glial tumor caused a marked behavioral disturbance, and Shy was demurring.  Just after I graduated, the Texas Tower shooting brought the question back up in force — https://en.wikipedia.org/wiki/University_of_Texas_tower_shooting.

Well that was 55 years ago, and we’ve learned a lot more about glia since.  

If glia don’t actually think, they may actually help neurons think better.  Since the brain is consuming 20% of your cardiac output as you sit there, it had better use the energy in the form of glucose  brought to it efficiently, and so it does, oxidizing it using oxygen (aerobic metabolism).  Glia on the other hand for reasons as yet unknown oxidize glucose anaerobically producing lactic acid (aerobic glycolysis). They transport the lactic acid to neurons and blocking transport impairs memory consolidation in experimental animals.  In fact aerobic glycolysis occurs in conditions of high synaptic plasticity and remodeling.  

The brain is 60% fat, some of which is cholesterol, which has to be made in the brain, as it doesn’t cross the blood brain barrier. Although neurons can synthesize cholesterol from scratch, most synthesis of cholesterol in the brain occurs in astrocytes.  It is than carried to neurons by apolipoprotein E.  As you are doubtless aware, apolipoprotein E (APOE) comes in three flavors 2, 3 and 4, and having two copies of APOE4 increases your risk of Alzheimer’s disease. 

But APOE does much more than schlep cholesterol to neurons according to a recent paper [ Neuron vol. 109 pp. 907 – 909, 957 – 970 ’21 ] Inside the particles are microRNAs.  You’ll recall that microRNAs decrease  the expression of proteins they target by binding to the messenger RNA (mRNA) for the targeted protein triggering its destruction. 

The microRNAs inside APOE suppress enzymes involved in de novo neuronal cholesterol biosynthesis (why work making cholesterol when the astrocyte is giving to you for free?).

This is unprecedented.  Passing metabolites (lactic acid, cholesterol) to neurons is one thing, but changing neuronal protein expression is quite another. 

Passing microRNAs in exosomes has been well worked out between cells (particularly cancer cells) outside the brain, but that’s for another time. 

Antioxidants — the dark side

There was (and probably still is) quite a vogue for antioxidants.  They were supposed to counteract aging, vascular disease, and prevent cancer.  So much so that 25 years ago, they were given in a trial to prevent lung cancer.  It didn’t work.  Here are the gory details

[ New England J. Med. vol. 330 pp. 1029 – 1035 ’94 ] The Alpha-Tocopherol, Beta-Carotene Trial (ATBC trial)  randomized double blind placebo controlled of daily supplementation with alpha-tocopherol (a form of vitamin E), beta carotene or both to see if it reduced the incidence of lung cancer was done in 29000 Finnish male smokers ages 50 – 69 (when most of the damage had been done).  They received either alpha tocopherol 50 mg/day, beta carotene 20 mg/day or both.   There was a high incidence of lung cancer (876/29000) during the 5 – 8 year period of followup.  Alpha tocopherol didn’t decrease the incidence of lung cancer, and there was a higher incidence among the men receiving beta carotene (by 18%).    Alpha tocopherol had no benefit on mortality (although there were more deaths from hemorrhagic stroke among the men receiving the supplement).   Total mortality was 8% higher among the participants on beta carotene (more deaths from lung cancer and ischemic heart disease).  It is unlikely that the dose was too low, since it was much higher than the estimated intake thought to be protective in the uncontrolled dietaryt studies.   The trial organizers were so baffled by the results that they even wondered whether the beta-carotene pills used in the study had become contaminated with some known carcinogen during the manufacturing process.  However, tests have ruled out that possibility.

Needless to say investigators in other beta carotene clinical trials (the Women’s Health Study, the Carotene and Retinoid Efficacy Trial) are upset.  [ Science vol. 264 pp. 501 – 502 ’94 ]  “In our heart of hearts, we don’t believe [ beta carotene is ] toxic”  says one researcher.

This is not science.

On to the present [ Cell vol. 178 pp. 265 – 267, 316 – 329, 330 – 345 ’19 ] in which the following appears “Recent evidence ‘suggests’ that antioxidants can also promote tumor formation”

The work concerns an animal model of nonsmallcell lung cancer (NSCLC).  I’m always wary of animal models, as they have been so useless in pointing to a useful therapy for stroke.  But the model is worth studying as it provides a mechanism by which antioxidants promote metastases of the primary tumor.  It is also worth studying because it shows the fiendish complexity of cellular biochemistry and physiology.

The only way you can appreciate complexity is by being buried in details. So let’s begin.  The actual details aren’t that important, just the number and the intricacy of them.

30% of humans with NSCLC have mutations in two genes (NFEL2L2, KEAP1).  The mutation in NFEL2L2 produces mutated NRF2 (a transcriptional activator of the antioxidant response gene set). The mutation doesn’t inactivate NRF2, but leaves it in a hyperactivated state.  KEAP1 normally inactivates NRF2, but not the mutated forms found in NSCLC.

One gene turned on by activated NRF2 is HO1 (heme oxidase).  During oxidative stress heme is released from heme containing resulting elevated intracellular heme lever resulting in the creation of free radicals which are inherently oxidative.  HO1 destroys heme. So this is one mechanisms of NRF2’s antioxidative activity.

Heme isn’t all bad, as it destabilizes BACH1 (not the composer)which is a prometastatic transcription factor.  Antioxidants (N-acetyl-cysteine, tocopherol [ vitamin E to you ] reduce heme levels stabilizing BACH1 (hence promoting metastasis).  Genes activated by BACH1 include glycolytic enzymes (hexokinase2, GAPDH).  So what?  Cancer cells use a lot of glycolytic enzymes even when oxygen is available — this is called aerobic glycolysis.  This is the Warburg effect.

I’m sure there’s far more to discover, but this should be enough to convince you that things are pretty complicated inside us.