Anti-aging properties of the aminosterols of the dogfish shark

Aging is associated with losses at many different levels, including physical activity, cognitive function, psychological and physical health, and resilience to stress¹. “Healthy aging” requires that we implement strategies to prevent, slow down, or effectively treat the most prevalent diseases of aging, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), Type 2 diabetes (T2D), cardiovascular disease, cancer, and infection. At a cellular level, diseases of aging are believed to result from the functional decline of homeostatic cellular processes within the nervous system, immune and hematopoietic tissues, cardiovascular and renal systems, liver, and GI tract. These cellular/physiological “hallmarks of aging”² are diverse and include: the basic cellular capacity to dispose of misfolded proteins and damaged organelles, a major problem for long-lived cells such as neurons and stem cells³; central insulin resistance, which underlies the pathophysiology of T2D and AD⁴; mitochondrial dysfunction resulting in impaired ATP synthesis and increased production of reactive oxygen species⁵; the reduced regenerative capacity of tissues and the accumulation of senescent cells⁶; chronic low-grade inflammation⁷; and progressive chromosomal alterations⁸.

The recognition that many processes are involved in the pathophysiology of diseases of aging presents a challenge for the discovery and development of drugs to extend the human healthspan. The traditional approach involves the discovery and development of drugs targeted at specific pathways, to be used in combination. A preferable approach would be the discovery of a single compound that by itself exhibited the desired pharmacodynamic health benefits through synergistic activity across the multiple interconnected networks involved in aging.

In 2002 the National Institute of Aging established a screening program (“Interventions Testing Program”) to evaluate the effect of treatments on the lifespan of the genetically heterogeneous UM-HET3 mouse stock⁹. Nine compounds have been identified to date: aspirin and nordihydroguaiaretic acid¹⁰, canagliflozin¹¹, captopril¹², glycine¹³, acarbose and Protandim¹⁴, 17α-estradiol¹⁵, and rapamycin^12,16,17. Of these, only rapamycin, administered at non-immunosuppressive doses, emerged as the most promising human clinical candidate in large part due to its efficacy in the mouse study in both genders, the overall clinical experience with the compound in organ transplantation, and relatively few adverse side effects at the proposed human doses. A second compound, metformin, widely used as a first-line treatment for Type 2 diabetes, is currently in Phase 3 clinical trials of life extension based on the results of longevity studies in rodents conducted in several laboratories¹⁸. It should be noted that metformin, except in combination with rapamycin, did not benefit lifespan in the NIA’s ITP study¹⁴.

Of all the well-studied (non-genetic) experimental interventions benefiting lifespan, caloric restriction appears to have the most dramatic impact, at least in rodents. Studies conducted in various mouse and rat strains have demonstrated that animals fed a diet that was calorically matched to 60% of that of an ad-lib fed control group exhibited a median lifespan between 30–50% longer than those provided food ad-lib, with minimal gender differences in the benefit¹⁹. We note in this context that trodusquemine exerts a dose-dependent reduction in food intake, discussed further below.

We discovered aminosterols in the liver of the dogfish shark (Squalus acanthias) in a search for anti-microbial compounds supporting the vertebrate innate immune system^20,21,22. This search began some years earlier with the discovery of anti-microbial peptides in the skin of the African Clawed frog (Xenopus laevis), motivated by the hypothesis that the surprising degree of immunity exhibited by certain animals could be due to the presence of simple anti-microbial compounds²³. The dogfish shark fell into that category. Sharks have an adaptive immune system that responds too slowly to defend itself against most bacterial or viral infections²⁴, so one might imagine that these animals would be relatively short-lived. Surprisingly, the dogfish enjoys a long, healthy lifespan, with an age limit of at least 100 years²⁵. The female becomes sexually mature at about 21 years, has the longest known gestation (about 2 years) of any animal^25,26, and delivers an average of about 6 offspring per cycle^27,28. Despite its small size at maturity (1–1.5 m, 5 kg), slow swimming speed²⁹, and low fecundity, the dogfish is the most abundant shark in the world, a fact that supports the conjecture that this species is characterized by a long healthy lifespan. Indeed, another member of the order Squaliformes, the Greenland shark (Somniosus microcephalus), is the longest-living vertebrate with a male lifespan of at least 392 ± 120 years, and female sexual maturity reached at 156 ± 22 years³⁰. Remarkably, a pathological examination of the brain of a 245-year-old female showed no signs of neurodegeneration³¹.

The physiological basis for the healthy lifespan of certain shark species remains unknown. However, as we will describe below, two aminosterols of the dogfish have been shown to target well-recognized aging-associated processes at both the cellular level, and in vivo in nematodes^32,33, zebrafish³⁴ rodents^{34,35,36,37,38,39,40,41,42,43,44,45,46}, dogs⁴⁷, humans⁴⁷, and horses^48,49,50,51. These observations, conducted in different laboratories, suggest that the dogfish aminosterols represent a novel class of endogenous vertebrate “geroprotectors.”

As a class, aminosterols are bioactive membrane lipids. Like the omega 3 fatty acid, docosahexaenoic acid (DHA), the most prominent essential membrane lipid in the brain⁵², aminosterols integrate directly into neuronal membranes, altering membrane fluidity, tensile strength, and lipid composition. The basic chemical structure of an aminosterol consists of a steroidal portion that resembles a bile acid or salt, and a polyamine (spermine or spermidine) coupled to the C3 of the steroid. Aminosterols with different biological activities have been discovered in certain species of shark^20,22, lamprey⁵³, and mouse⁵⁴, each differing with respect to the polyamine and the functionalities occupying C24, C26, and C27. The most extensively studied aminosterols are trodusquemine and squalamine (Fig. 1). At physiological pH, both aminosterols exist as a zwitterion with a net cationic charge. As a consequence, the aminosterol binds electrostatically to membranes containing negatively charged phospholipid and glycolipid headgroups. Upon binding, it buries itself partially within the lipid bilayer. The neutralization of the negative charge on the membrane surface makes it less attractive to cationic proteins^33,55,56. The affinity of cholesterol and anionic phospholipids for the cationic aminosterol induces the spatial reorganization of the lipid composition of the membrane⁵⁷. As a consequence, both the tensile strength of the membrane (the force required to puncture the membrane) as well as its fluidity are increased^58,59. As would be expected, the magnitude of these effects is proportional to the concentration of trodusquemine within the membrane (Fig. 2).

The structures are presented as the zwitterions that exist at physiological pH.

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The system illustrated is described in ref. ⁵⁴, from which the Figure has been modified. A Trodusquemine is attracted electrostatically to the negatively charged GM1 ganglioside and subsequently buries itself in the membrane, as illustrated. The overall negative surface charge of the membrane is reduced due to ion-pairing. As a consequence, amphipathic cationic proteins bound to the membrane are displaced. B The spatial organization of lipids within the membrane is changed. Phospholipids with negatively charged headgroups cluster around the trodusquemine molecules, and cholesterol-rich domains segregate. The tensile strength of the membrane is increased. By this biophysical mechanism trodusquemine appears to defend a biological membrane from the toxic action of misfolded proteins.

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Studies conducted in vitro, both with synthetic neuronal membranes and with cultured neuronal cells, demonstrate that in the presence of either squalamine or trodusquemine, cytotoxic aggregates of misfolded proteins are both displaced from the membranes and prevented from binding. Proteins that have been studied include α-synuclein, Aβ₄₀ Aβ₄₂, TDP-43, and a cytotoxic amyloidogenic E. coli protein HypF-N^{32,33,54,60,61,62,63,64}. These studies permit us to conclude that misfolded proteins that exhibit cellular toxicity display both hydrophobic and cationic amino acids on their surface, properties that support their binding to cellular membranes. We assume that only a subset of misfolded proteins that cause disease are known. If so, the protective effects of trodusquemine and related aminosterols would represent a general defense against misfolded proteins. Since it appears that intracellular misfolded proteins (as defined by protease resistance) increase with natural aging and cellular senescence⁶⁵, it would make sense to evaluate the cellular effects of aminosterols in aging-associated human diseases.

Although trodusquemine and squalamine share similar biophysical properties in vitro, they exhibit different pharmacological activities in vivo due to the impact of the specific polyamine (spermine vs spermidine) on biodistribution and, as a consequence, its pharmacodynamic profile. In particular, while systemically administered trodusquemine accesses the brain, squalamine does not. Recent studies with squalamine have focused on its effects on the enteric nervous system (ENS)^66,67,68. We conducted two highly successful Phase 2 trials involving over 200 elderly patients with PD, which showed that a brief, 3-week course of ENT-01 (squalamine phosphate) normalized circadian rhythm, reversed constipation, and significantly improved sleep, hallucinations, dementia, and depression^69,70. ENT-01 is administered orally, crosses the gut epithelium, is incorporated into the enteric neuronal membrane, displaces aggregated alpha-synuclein, the pathologic misfolded protein responsible for PD, and restores enteric neuronal function, which includes vagal gut-brain communication. Orally administered squalamine is not absorbed into the systemic circulation, so its pharmacological effects are due to local action on the gastrointestinal tract. Electrophysiological studies in wild type mice demonstrate that vagal neural signals from gut to brain diminish in amplitude and frequency with aging; oral administration restores the vagal signals of the elderly mouse to that of the young animal, likely due to displacement of misfolded proteins within neurons of the ENS^68,71. If the ENS of the aging human suffers the same pathophysiology as the aging mouse, oral administration of squalamine could impact bowel dysmotility (common in the elderly) and potentially also sleep architecture, depression, and cognition, as observed in the clinical trials with a PD population.

The effects of trodusquemine have been studied in great detail at both the cellular level, and in vivo and will be the focus of the remainder of the review. From many studies, we can construct a cellular-based mechanism of action for trodusquemine that helps explain the diverse pharmacological activities of this compound and its potential therapeutic applications (Table 1).

The cellular accumulation of misfolded proteins and the resultant effects on cellular function, such as ER stress, are well-recognized components of the pathophysiology of the diseases of aging^65,72. In each of these conditions, cells within the brain or peripheral organs produce excessive amounts of protein, whether it be a specific molecule, such as Tau in AD, alpha-synuclein (αS) in PD, or a large constellation as in Type 2 diabetes and obesity⁷³, or the proteins of an infectious agent such as a virus^74,75. Proteins that fail to adopt their tertiary structure are normally cleared by a complex cellular process, the unfolded protein response (UPR). As misfolded proteins accumulate within the cell, the UPR signals the cell to slow down protein synthesis, and increase the activity of the cellular protein garbage disposal machinery (lysosomes, autophagy).

When the UPR is overwhelmed, the cell experiences “ER stress.” Membrane-disruptive misfolded protein aggregates accumulate within the cell to toxic concentrations. Functionally “out-of-date” mitochondria, leaking reactive oxygen species and producing reduced amounts of ATP, are no longer effectively removed by autophagy and are replaced. Lysosomes “clog-up,” as their membrane systems are damaged. The UPR becomes a “suicidal” pathway, ultimately driving cellular death. The central nervous system is at a particular disadvantage to ER stress since many of its cells are present for a lifetime, and the death of a cell will not be followed by a replacement. Since common diseases of aging such as dementia, AD, PD, cardiovascular disease, T2D/obesity, and susceptibility to infectious disease are associated, at the cellular level, with ER stress, it would appear that as we age our tissues become prone to ER stress. Why this should be a consequence of aging remains unknown.

When a neuronal cell is exposed to trodusquemine the molecule initially binds to the plasma membrane, and then over the course of several hours, it circulates within the membrane system of the neuron, progressively moving from its site of entry at the plasma membrane to the deeper intracellular membrane compartments such as the endoplasmic reticulum (ER) and the Golgi apparatus, and eventually lysosomal membranes⁷⁶. As a consequence, aggregates of misfolded proteins are displaced from intracellular membranes, ER stress is relieved^{44,48,49,50,51} lysosomal activity is restored, as are autophagic activity^48,49,50,51, and mitochondrial health⁴⁵. The various effects of trodusquemine at a cellular level are summarized in the diagram in Fig. 3.

Left panel: Intracellular misfolded proteins are shown to bind to cellular membranes, resulting in organelle dysfunction and ER stress. Intracellular membranes rich in anionic phospholipids preferentially attract misfolded proteins. PTP1B activity is increased, impairing insulin signaling. Right panel: Trodusquemine, an amphipathic cationic zwitterion, integrates into cell membranes and displaces membrane-bound misfolded proteins electrostatically, restoring normal organelle functions. Inhibition of PTP1B restores normal insulin signaling.

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Within the context of the UPR, the enzyme PTP1B is of particular interest to us. PTP1B is a membrane-associated tyrosine phosphatase that plays a major role in the regulation of the activity of many receptor tyrosine kinases that utilize the PI3K/AKT pathway, such as insulin and IGF1, and the JAK-Stat pathway, such as the neurotrophins and ciliary neurotrophic factor (CNTF). PTP1B turns off circuits by removing phosphate from the activated receptor or downstream phosphorylated proteins. In cells that are experiencing ER stress, the activity of PTP1B is abnormally elevated⁷⁷. Inhibition of PTP1B curbs ER stress with diseases as diverse as metabolic syndrome or atherosclerosis. In obese diabetic animals, supra-normal concentrations of insulin are required to activate the insulin pathway, resulting in insulin resistance. Inhibition of central PTP1B activity restores the animal’s insulin sensitivity.

Trodusquemine is the most extensively studied inhibitor of PTP1B, with a large body of research conducted in animals that appears to support the targeting of PTP1B in different disease states (Table 1). Trodusquemine inhibits PTP1B through a well-characterized allosteric mechanism^78,79. However, trodusquemine also inhibits other non-receptor tyrosine phosphatases with comparable potency, such as the activated form of PTPN11 (SHP2) (Zasloff, unpublished), the dopamine and norepinephrine transporters⁴¹, and the NMDA receptor (Chiti et al. manuscript submitted).

Early on, we discovered that trodusquemine caused obese diabetic rodents to reduce food intake, lose adipose tissue, restore insulin sensitivity, normalize metabolic parameters, including blood glucose, and reverse hepatic steatosis^36,37,38,41. Since these effects could be observed by either systemic or intra-cerebroventicular administration of much smaller amounts of the compound, we concluded that trodusquemine acted via a central mechanism^36,37. Autoradiographic studies in mice administered tritiated trodusquemine confirmed that the compound accessed the brain and localized to specific areas of the brain, such as the hypothalamus and the arcuate nucleus, which are involved in the regulation of energy homeostasis, diurnal rhythm, and a multitude of other physiological functions. Trodusquemine inhibits PTP1B within neurons of the mediobasal hypothalamus, resulting in an increase in the response of the brain to both insulin and leptin. The “classical” in vivo assay for the activity of trodusquemine or an analog is a drug-induced, dose-dependent reduction in caloric intake, weight loss with subsequent gradual recovery of weight following cessation of treatment^36,37,41.

Trodusquemine has been studied in the equine metabolic syndrome, a syndrome similar to the human metabolic syndrome^49,50. The condition arises due to inadequate physical activity and excessive consumption of carbohydrates. Symptoms include insulin resistance, hyperlipidemia, chronic low-grade inflammation, and progressive liver disease associated with inflammation, and fibrosis. Horses were administered a single, very low dose of trodusquemine (25 µg/kg, iv), and liver biopsies and fasting blood were studied one month post treatment. The single dose caused a reduction in blood insulin and glucose. Liver biopsies from the treated horses demonstrated reduced expression of components of the UPR (CHOP, GRP78 PERK, and ATF6) along with PARKIN and PINK1, reflecting normalization of mitochondrial damage and ER stress. Circulating proinflammatory cytokines, which were substantially lowered in treated animals, included IL-1β, TGFβ, and TNFα, along with decreases in expression of their corresponding transcripts from liver biopsies. Significant increases in circulating regulatory T cells and reduction in white blood cells/monocytes were observed with treatment. This study, (along with companion reports) provides the most comprehensive evidence in vivo that trodusquemine has the potential to correct pathological ER stress and mitochondrial dysfunction in the liver and reduce systemic inflammation. It is conceivable that the effects of trodusquemine on liver health result from direct action on the liver. However, it is known that reduction of ER stress within the hypothalamus can indirectly lower ER stress in the liver via autonomic signals, raising the possibility that the beneficial effects of trodusquemine on peripheral organs are centrally mediated⁸⁰.

Trodusquemine was evaluated for T2D and obesity in three Phase 1 clinical trials involving a total of 71 patients⁴⁷. The compound was administered as an iv infusion over 30 minutes at doses ranging from 3–30 mg/m². Dose-dependent reduction in blood sugar, insulin, and body weight was observed following either single or multiple doses. The principal adverse reactions across the dosing range were infusion site pain and local venous inflammation. Attempts to reformulate the compound to permit administration by other routes were not performed, leading to the discontinuation of the development program. These clinical studies, along with the human pharmacokinetic data, provided sufficient evidence that the preclinical pharmacology of trodusquemine on insulin resistance could be translated to humans.

Chronic treatment with trodusquemine of low-density lipoprotein receptor knock-out mice (LDLR^-/-) fed on a high-fat diet (HFD) attenuated aortic atherosclerotic plaque formation. A single dose significantly reversed pre-existing plaque. The aortic tissues of treated animals exhibited markedly increased phosphorylation (activation) of AMPK as a consequence of the inhibition of PTP1B, leading to reduced local inflammation, clearance of cholesterol, and enhanced lipid oxidation³⁹.

Calcific aortic valvular disease (CAVD) has become more prevalent as the population ages. Lipid-lowering drugs such as simvastatin do not appear to prevent CAVD. The discovery (above) that inhibition of PTP1B protected against atherosclerotic plaque formation inspired the subsequent discovery that PTP1B activity was markedly elevated in tissues from calcific human aortic valves⁴⁵. Aortic valves from LDLR^-/- mice, fed on an HFD express abnormally high concentrations of PTP1B when examined by immunofluorescence, and aortic stenosis and valvular calcification develop over a 24-week period. In contrast, once monthly systemic administration of trodusquemine, using a regimen that has no effect on body weight, completely prevents CAVD. In animals fed an HFD, valvular tissues from untreated animals exhibited depleted numbers of mitochondria, while mitochondrial numbers were preserved in valvular tissues from the treated cohort. This study, which identifies PTP1B as a regulator of mitochondrial health in arteries and valves subjected to flow-based trauma, positions trodusquemine as a potential therapeutic or prophylactic in the setting of the aging cardiovascular system.

Vascular endothelial dysfunction occurs during aging and plays a key role in the pathophysiology of cardiovascular and cerebrovascular disease. In small vessels, endothelial dysfunction is characterized by reduced vascular dilatation in response to increased intravascular pressure. In particular, vascular endothelial cells lose their capacity to produce the potent vasodilator, nitric oxide. Recent studies have demonstrated that in mouse models of diabetes, sepsis, and heart failure, vascular endothelial cells are in a state of ER stress. Since genetic knock-out of PTP1B restored vascular relaxation and endothelial eNOS phosphorylation with therapeutic benefit in these disease models, treatment with trodusquemine was subsequently studied⁴⁴. Small resistance arteries of wild-type mice were treated ex vivo with tunicamycin to induce ER stress. As expected, these vessels failed to dilate in response to increased pressure, compared with vessels not exposed to tunicamycin. When trodusquemine was introduced along with tunicamycin, the arteries dilated as pressure increased. In a parallel experiment, tunicamycin had no effect on arteries from PTP1B knock-out mice, consistent with the PTP1B inhibitory effect of trodusquemine in this model. These studies suggest a role for trodusquemine in both prevention and treatment of age-related vascular disease.

Regenerative capacity in organs and tissues diminishes with age. The discovery of small molecules that can safely restore regenerative capacity lost during aging would have great therapeutic value in many age-related diseases. Trodusquemine was discovered to have potent regenerative properties in an unbiased screen of diverse compounds using the zebrafish tail amputation model³⁴. Treatment of adult zebrafish with trodusquemine stimulated the rate of regeneration of caudal fin tissue and heart muscle by 2–3-fold without apparent tissue overgrowth or malformation. Inhibition of PTP1B by treatment of the zebrafish with antisense RNA stimulated cardiac regeneration to the same extent as trodusquemine, supporting the hypothesis that PTP1B was the primary target. Furthermore, administration of trodusquemine to adult mice for 4 weeks beginning 24 h after the induction of cardiac ischemia by coronary artery ligation increased survival, improved ejection function ~2-fold, reduced infarct size by 53%, reduced ventricular wall thinning and increased stem cell proliferation in the infarct border zone ~4-fold relative to vehicle-treated animals. Lastly, treatment of mice with trodusquemine 24 hours after skeletal muscle injury stimulated satellite cell activation ~2-fold. This study is consistent with an earlier report that genetic deletion of PTP1B in mice accelerates hepatic regeneration in the setting of fatty liver disease associated with a HFD⁸¹. A plausible mechanism to explain Trodusquemine’s ability to stimulate the regeneration of an entire complex structure, such as a caudal fin or heart, focuses on its ability to increase the activity of numerous growth factor receptors. Conceptually, as the regenerative process unfolds, successive waves of diverse growth factors and their receptors are expressed in an exquisitely orchestrated pattern. The inhibition of PTP1B may simply enhance the gain on the regenerative circuits without disturbing the sequence of events. Regeneration of the zebrafish tail, which is comprised of bone, connective tissue, skin, vascular and nervous tissues, like amphibian limbs, must require neural input. This opens the possibility that trodusquemine, which crosses the blood brain barrier and localizes to the mediobasal hypothalamus and floor of the 3rd ventricle could be directing the regenerative processes by a central mechanism. This study suggests that trodusquemine has the potential to restore regenerative capacity lost with aging to many organs, including the brain.

Aging-associated changes to the immune system are clinically important, making older individuals more vulnerable to infection and cancer. A reduced number of naive circulating CD8 + T cells is the most consistent and prominent marker of immune senescence^82,83. Cytotoxic CD8 + T cells play a major role in the immune defense against intracellular infections and malignant cells. PTP1B has recently been shown to negatively regulate the anti-tumor efficacy of cytotoxic T cells⁴⁶. Both global deletion of PTP1B and specific deletion within the T-cell compartment enhanced the proliferation, activation, cytotoxicity, and anti-tumor activity of CD8 + T cells. Mechanistically, PTP1B appears to down regulate IL-2 promoted T-cell proliferation, survival, differentiation, and cytotoxicity. Based on these observations, trodusquemine was administered to mice bearing tumor allografts. Trodusquemine markedly suppressed tumor growth accompanied by the infiltration of effector CD8 + T cells, memory CD8 + T cells, natural killer cells, dendritic cells, and tumor-associated macrophages. In addition, trodusquemine administration caused an increase in naïve T cells in the spleen and activated naïve, effector, and memory T cells in lymphoid organs. This study suggests that trodusquemine has the potential to restore immune competence against infection and cancer that diminish with aging.

We have conducted several studies on the effects of trodusquemine and closely related aminosterols in two mouse models of AD. The results of these studies demonstrate remarkable and robust improvement in all core pathological and behavioral features of the disease⁸⁴.

Neuroinflammation and neurodegeneration are common features of familial and sporadic AD. However, it is not clear whether neuroinflammation or neuronal cellular dysfunction per se account for cognitive dysfunction and neurodegeneration. Neuroinflammation and ER stress, both associated with amyloidosis and commonly observed in AD^85,86,87, result in increased activity of the protein tyrosine phosphatase 1B (PTP1B)^88,89,90.

Repeated administration of trodusquemine (5 mg/kg, i.p., every 5 days for 6 doses) improved spatial memory in hAPP-J20 mice in the Morris water maze test (p < 0.001). HAPP-J20 mice are reported to exhibit elevated degrees of inflammation, which may contribute to cerebral deterioration and cognitive dysfunction^91,92. Activation of innate immune cells (microglia), as shown by IBA immunostaining was detected in the hippocampus CA3 region of hAPP-J20 mice. In addition, increased gliosis was observed by immunostaining for the astrocyte marker GFAP-positive area compared with vehicle-treated WT mice. We observed inflammation before the accumulation of amyloid protein and plaque deposition in hAPP-J20 mice, suggesting that inflammation is an early response in this AD model. Trodusquemine treatment prevented an increase in the IBA1-positive area in hAPP-J20 mice, implying that trodusquemine attenuates microgliosis in hAPP-J20 mice. Similarly, the GFAP-positive area was greater in vehicle-treated hAPP-J20 mice compared with vehicle-treated WT mice and was markedly reduced by trodusquemine treatment.

HAPP-J20 mice display aging-associated neurodegeneration in the hippocampus⁹¹. To determine whether PTP1B inhibition affects neuron loss to account for its effect on preserving cognitive function in hAPP-J20 mice, we compared neuronal numbers by quantifying NeuN-positive cells in the pyramidal cell layer of hippocampal regions. Vehicle-treated hAPP J20 contained significantly fewer neurons in the CA3 region, compared with WT mice. This neuron loss was prevented when hAPP-J20 mice were treated with trodusquemine.

In a related study, we examined the effect of trodusquemine on synaptic plasticity within the CA3 region of the hippocampus of hAPP J20 mice using electrophysiological methods. hAPP J20 mice are known to develop deficits in long term potentiation (LTP) within the hippocampus⁹³. In our study, using whole cell patch-clamp recording, we demonstrated that hAPP J20 mice exhibit a profound presynaptic deficit in LTP of CA3:CA1 synapses which impairs spatial learning. This deficit can be corrected by treatment with trodusquemine.

In summary, we show that treatment of the hAPP-J20 familial AD mouse model with trodusquemine prevents neuronal loss, reduces inflammation, and improves memory. It remains uncertain the extent to which other potential benefits of trodusquemine in this model such as improved cerebral blood flow and increased insulin sensitivity influence the observed improvement.

Rapamycin has been shown in repeated independent studies to increase the lifespan of male and female mice⁹⁴. As yet, no studies of life prolongation by trodusquemine in mice have been published, but we can compare the two compounds with respect to their known effects on the mechanisms underlying aging-associated diseases.

Both are endogenous natural products that exhibit potent anti-microbial activity, suggesting that the selection pressure driving their evolution was a defense against micro-organisms, an essential survival function. Their mechanisms of anti-microbial action are profoundly different but surprisingly explain their activity in mammalian systems. Rapamycin was shown to inhibit fungal growth by targeting an intracellular protein named “TOR”⁹⁵; an orthologous protein target, “mTOR” was shown to be the target of the compound in mammalian cells^96,97,98. The fundamental targets of trodusquemine, in contrast, are intracellular membranes. In the case of bacteria, which display anionic phospholipids on the external leaflet of their plasma membrane, the aminosterols irreversibly bind through electrostatic interactions and disrupt membrane integrity^99,100. Mammalian cells, in contrast, segregate membranes rich in anionic phospholipids inside the cell¹⁰¹. Aminosterols, like trodusquemine, first must enter a mammalian cell, then find their way onto specific membrane surfaces with the appropriate surface density and spatial organization of anionic phospholipids to impact cellular activities.

Both rapamycin and trodusquemine induce clearance of misfolded proteins and attenuate ER stress. Rapamycin does so by inhibiting mTORC1, which then signals the cell that nutrients are in short supply, cell growth must stop, and lysosomal and autophagic activities must be ramped up to recycle non-essential proteins and lipids¹⁰². Trodusquemine achieves the same end with respect to ER stress through electrostatic displacement of misfolded proteins from intracellular sites and inhibition of PTP1B, as described above.

Since malignancy is the major aging-related cause of death in mice^103,104,105, the anti-cancer effects of rapamycin weigh heavily on its impact on longevity in these animals⁹⁴. Both rapamycin and, as noted previously, trodusquemine, exhibit anti-cancer activity in vivo. Rapamycin is believed to control malignancy via its effects on cell growth and proliferation, and the apparent increase in mTORC1 activity in many types of cancer¹⁰⁶. In contrast, trodusquemine exerts its anti-cancer activity either through direct effect on the tumor cells and /or through inhibition of PTP1B in immune cells resulting in the mobilization of cytotoxic T cells⁴⁶.

The effects of rapamycin on obesity, diabetes and their cardiovascular complications present a complex picture. Rapamycin, like trodusquemine, has been shown to reduce atheroma in LDLR-/- mice fed a high-fat diet⁹⁴. However, unlike trodusquemine, which restores plasma cholesterol and triglycerides to normal, rapamycin has minimal effects on these parameters, and in humans increases both⁹⁴.

Like trodusquemine, rapamycin has been shown to prevent weight gain in mice and rats fed high-fat diets associated with reduced fat mass, possibly due to inhibition of lipogenesis and hepatic steatosis^{107,108,109,110}. However, while trodusquemine has been shown to correct insulin resistance and correct hyperglycemia, extended treatment with rapamycin, in contrast, appears to cause insulin resistance, hyperglycemia, and glucose intolerance¹¹⁰. The physiological mechanism of rapamycin’s effect involves suppression of insulin-dependent inhibition of hepatic gluconeogenesis, possibly involving inhibition of mTORC2¹¹¹ and reduced glucose uptake by skeletal muscle¹⁰⁷. Trodusquemine has been shown to effectively reverse rapamycin-induced diabetes, through its inhibitory effects on hepatic gluconeogenesis¹¹². Everolimus, a rapamycin analog, has been shown to improve cardiac output following experimental myocardial infarction in rodents but appears to achieve this effect through different mechanisms than trodusquemine. In the case of Everolimus, treatment benefit in a rat model appeared to derive from increased autophagic clearance of damaged tissue within the infarction zone resulting in a reduction in infarct size and inhibition of left ventricular remodeling¹¹³. Trodusquemine also reduced infarct size and improved ejection fraction in a mouse model, but most significantly stimulated mobilization of cardiac muscle stem cells within the infarct margins leading to the formation of new cardiac muscle³⁴.

Lastly, rapamycin has been shown to have beneficial effects in mouse models of AD, PD, and Huntington’s disease, as well as other neurodegenerative conditions⁹⁴. Although the mechanisms remain to be defined, like trodusquemine, rapamycin’s property of increasing the elimination of intracellular misfolded proteins is likely involved. Indeed, such a mechanism was invoked to explain the stimulatory effect of rapamycin on neurogenesis in the aging mouse brain¹¹⁴.

When should treatment with an anti-aging drug begin? What additional benefit might one derive by initiating treatment in individuals with significant risk factors, prior to disease onset, such as is our current practice in the treatment of hypertension and hyperlipidemia? In addition, what further benefit might accrue should the treatment with anti-aging agents begin in early childhood? The period of postnatal development is regarded as a critical stage during an organism’s lifetime due to its influence on the future trajectory for health and vitality^115,116,117. Previous studies in rodents have shown that early life interventions such as rapamycin treatment, growth hormone replacement therapy, food restriction, and manipulation of litter size have significant effects on growth rate, age of sexual maturity, and mortality^{118,119,120,121,122,123,124,125}. The goal should be not only increasing lifespan but also proactively forestalling age-related diseases.

We have recently compared the metabolic effects of trodusquemine with metformin in neonatal UM-HET3 mice treated from the age of 2 weeks through 8 weeks¹²⁶. Metformin was used because its metabolic effects mimic calorie-restriction¹²⁷ in part through activation of the hepatic cellular energy sensor AMPK, a key regulator of lipid and glucose catabolism¹²⁸. While both metformin and trodusquemine improved glucose homeostasis, trodusquemine alone significantly affected an “anti-aging” transcriptional phenotype in the livers of both male and female juvenile UM HET mice not unlike that expected from caloric restriction: the inhibition of genes involved in growth and energy metabolism (Pi3k, Akt2, mTOR, Srebp1), fatty acid uptake and lipogenesis (Cd36, Acc, Scd1), energy sensing (Sirt1), cholesterol transport and homeostasis (Apob, Apoe, Apoa1, Apoa4, Lxr, Abcg5, Cyp7a1) and VLDL-transport (Cideb, Mttp, Sar1a, Sar1b, Sec22b, Stx5a). The impact of the administration of trodusquemine on the health trajectory of a neonatal or juvenile subject is currently being investigated.

We have not yet conducted formal lifespan studies with trodusquemine in any species. A definitive statement to the effect that trodusquemine extends lifespan cannot, therefore, yet be made. However, the dramatic effects of trodusquemine on all age-related conditions described above would be expected to increase the healthy human lifespan. Studies are currently being conducted in animals with age-related diseases with the intention of extending studies to humans. Squalamine (ENT-01) has completed Phase 2 clinical trials in PD and is poised to begin Phase 3 evaluation.

We have shown that trodusquemine targets several major age-related conditions, suggesting that this compound and related aminosterols have the potential to extend the healthy human lifespan. We favor endogenous compounds with anti-aging properties that have evolved over hundreds of millions of years within the biological context of long-lived animals over man-made compounds targeting single pathways. As we embark on the development of pharmaceutical agents to target diseases of aging, the question arises as to whether such an agent could also extend the biological limits of the lifespan. Since the biological basis of “evolution’s iron law¹²⁹” is unknown, one cannot discount the possibility that the upper range of the human lifespan could be extended significantly. Although we naturally think of life extension, in a philosophical sense, as exceedingly complex, potential solutions could be straightforward, such as the gradual decline with age in the expression of an endogenous compound. The potential of the dogfish aminosterols as anti-aging drugs await their evaluation in appropriate human clinical trials.