Huntington Potter

Previous studies showed that granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment reduced cerebral amyloidosis, cognitive impairment, and neuronal death in animal models of Alzheimer’s disease (AD), and improved MMSE scores and plasma measures of AD neuropathology and neuronal death in a Phase 2 trial. Type 2 diabetes (T2D) is characterized by pancreatic amyloid deposits of human islet amyloid polypeptide (hIAPP), islet b- and a-cell apoptosis, and elevated blood glucose levels. We report that GM-CSF treatment of hIAPP transgenic male mice decreased pancreatic amyloid (P=0.0140) and decreased islet apoptosis (P=0.0057) compared to saline-treated mice. GM-CSF treatment also led to a sustained decrease in pancreatic islet amyloid at 45 days post-treatment (DPT) in homozygous hIAPP mice (P=0.045; especially in males, P=0.0214) and reduced blood glucose levels from baseline through 45 DPT (P=0.0002 compared to saline treatment). Recombinant human GM-CSF (sargramostim), long-approved for other indications, may prevent/treat T2D and potentially reduce AD associated with T2D.

Down syndrome (DS) is associated with a myriad of cardiovascular defects. Endothelial cells, which form the innermost layer of blood vessels, have been found to exhibit divergent morphology and functional impairments in the peripheral circulation of individuals with DS. DS is also a strong risk factor for Alzheimer's disease‐related neuropathology. Recent evidence linking cerebral vasculature to Alzheimer's disease, along with established correlations between cardiovascular and cognitive functions, prompted us to investigate brain parenchymal microcirculation reactivity in a DS mouse model. We used brain parenchymal arterioles to investigate the impact of DS on endothelium‐dependent vasodilatation using the Dp16 mouse model coupled with ex vivo pressure myography and in vivo two‐photon laser‐scanning microscopy. Two age cohorts of mice, young and middle‐aged adults, were employed to test for age dependence, with the imminent development of Alzheimer's disease with age of individuals with DS in mind. Using pressure myography combined with pharmacological inhibitors, we examined two essential vasodilatory pathways in intracerebral arterioles – endothelial nitric oxide synthase and endothelium‐dependent hyperpolarization mediated by small‐ and intermediate‐conductance potassium channels. These pathways exhibited reduced constitutive activity in the DS model in both age groups. Consistent with these impairments, we measured higher levels of pressure‐induced constriction, also referred to as myogenic tone, in arterioles from the cortex of Dp16 mice seen both in vivo and ex vivo . Together, our findings demonstrate altered cerebrovascular functionality in DS and identify possible therapeutic targets for alleviating symptoms associated with DS. image Key points: Down syndrome is associated with cardiovascular diseases. Endothelium‐dependent hyperpolarization and nitric oxide synthase activity are reduced in Dp16 brain parenchymal arterioles. Parenchymal arteriole myogenic tone is elevated in Dp16 mice both ex vivo and in vivo . Endothelial dysfunction is a therapeutic target in Down syndrome. Restoration of cerebral blood flow through vascular modulation may be beneficial.

Background: Increasing age is the greatest risk factor for developing Alzheimer's disease (AD) and of ‘normal’ cognitive decline. People with Down syndrome/trisomy 21 (DS) have cognitive challenges throughout life and have a near 100% risk of developing AD neuropathology by age 40, with most developing AD dementia by age 60. We showed in a cross‐sectional study of community dwelling normosomic individuals that plasma concentrations of UCH‐L1 and NfL, measures of the rate of neuronal cell loss/apoptosis and axon damage, respectively, show exponential increases with age starting in early childhood. Plasma concentrations of GFAP, a marker of astrogliosis/inflammation, increase exponentially starting at around age 40. We also showed that the immune system modulating cytokine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) improved cognition and reduced neuronal apoptosis in animal models of aging, AD, and DS, and that treatment of AD participants with human recombinant GM‐CSF/sargramostim in a phase II trial improved a measure of cognition, reduced plasma UCH‐L1, and partly normalized plasma Ab40 and total Tau (PMID:39072024). Method: Plasma concentrations of UCH‐L1, NfL, and GFAP were assessed in 316 individuals with DS age 2‐60 using the Quanterix SIMOA ^Ò platform and compared to previous findings from normosomic, healthy, age‐matched controls. Neuronal apoptosis in the Dp16 mouse model of DS was assessed by Caspase‐3 staining. Result: Plasma concentrations of biomarkers of ongoing neuronal damage (UCH‐L1 and NfL) and gliosis/inflammation (GFAP) in the brain are significantly increased in individuals with DS starting in childhood, and their exponential increase with age occurs at a much faster rate than in normosomic individuals. Preliminary measures of neuronal apoptosis and gliosis are also increased in the brains of Dp16 mice. Conclusion: Age‐associated increases in plasma measures of the rate of neuronal death/damage and astrogliosis/inflammation in people with DS compared to normosomic individuals and of neuronal apoptosis in a mouse model of DS indicate an accelerated rate of brain aging, likely caused by increased APP expression and consequent production of neurotoxic Ab and exacerbated by astrogliosis/inflammation. Clinical trials are indicated for testing whether GM‐CSF (sargramostim) treatment may reduce neuronal death/damage and gliosis and improve cognition in young adults with DS and in typical normosomic aging individuals.

Background: Increasing age is the greatest risk factor for age‐associated cognitive decline (AACD) and, especially in females, for developing Alzheimer’s disease (AD). Mechanisms underlying this connection are unknown, but neuronal loss and brain atrophy accompany aging and AD and likely contribute to cognitive deficits. There are currently no means to measure neuronal cell death during life and no means to prevent it. Inflammation is also correlated with AD pathogenesis and aging (‘inflammaging’), but whether inflammation causes and/or is a response to neurodegeneration is unknown.Treatment of mouse models of AD with GM‐CSF reverses amyloid deposition and normalizes cognition, and GM‐CSF treatment improves cognition in aged wild‐type mice. A published double‐blind placebo‐controlled phase II trial showed that three weeks of treatment with recombinant human GM‐CSF (sargramostim) in mild‐to‐moderate AD participants improved cognition (MMSE) and blood biomarkers of amyloid, Tau, and neurodegeneration. Together, these results suggest that GM‐CSF may slow or halt the neuronal damage in aging and/or AD that causes cognitive deficits.Method: Plasma concentrations of ubiquitin C‐terminal hydrolase‐L1, UCH‐L1, a measure of neuronal cell loss, neurofilament light (NfL), a measure of axonal damage, and GFAP, a measure of inflammation/astrogliosis, were assessed cross‐sectionally in 317 healthy control participants between age 2 and 85 using the Quanterix SIMOA platform. The resulting age curves were compared to the results from the previously published clinical trial of sargramostim/GM‐CSF in participants with mild‐to‐moderate AD.Result: The concentrations of human plasma proteins released from dying/damaged neurons (UCH‐L1 and NfL) showed an exponential increase from age 2‐85 and rise more in females. Plasma GFAP concentrations are exponentially higher after age 40, following neurodegeneration. Treatment of AD participants in the GM‐CSF/sargramostim trial halted neuronal cell death, based on the reduction in plasma UCH‐L1 concentrations to levels equivalent to those of five‐year‐old healthy controls. The ability of GM‐CSF treatment to reduce neuronal apoptosis was confirmed in the hippocampi of aged TgF344‐AD rats, a model recapitulates all human AD brain neuropathology.Conclusion: These findings suggest that age‐associated exponential increases in neurodegeneration may underlie the contribution of aging to cognitive decline, including in AD, and that GM‐CSF/sargramostim treatment may halt these effects.

Summary: Aging increases the risk of neurodegeneration, cognitive decline, and Alzheimer’s disease (AD). Currently no means exist to measure neuronal cell death during life or to prevent it. Here we show that cross-sectional measures of human plasma proteins released from dying/damaged neurons (ubiquitin C-terminal hydrolase-L1/UCH-L1 and neurofilament light/NfL) become exponentially higher from age 2-85; UCH-L1 rises faster in females. Glial fibrillary acidic protein (GFAP) concentrations, indicating astrogliosis/inflammation, increase exponentially after age 40. Treatment with human granulocyte-macrophage colony-stimulating factor (GM-CSF/sargramostim) halted neuronal cell death, as evidenced by reduced plasma UCH-L1 concentrations, in AD participants to levels equivalent to those of five-year-old healthy controls. The ability of GM-CSF treatment to reduce neuronal apoptosis was confirmed in a rat model of AD. These findings suggest that the exponential increase in neurodegeneration with age, accelerated by neuroinflammation, may underlie the contribution of aging to cognitive decline and AD and can be halted by GM-CSF/sargramostim treatment.

Adult-onset diabetes increases one’s risk of neurodegenerative disease including Alzheimer’s disease (AD); however, the risk associated with youth-onset diabetes (Y-DM) remains underexplored. We quantified plasma biomarkers of neurodegeneration and AD in participants with Y-DM from the SEARCH cohort at adolescence and young adulthood (Type 1, n = 25; Type 2, n = 25; 59% female; adolescence, age = 15 y/o [2.6]; adulthood, age = 27.4 y/o [2.2]), comparing them with controls (adolescence, n = 25, age = 14.8 y/o [2.7]; adulthood, n = 21, age = 24.9 y/o [2.8]). Plasma biomarkers, including glial fibrillary acidic protein (GFAP), neurofilament light chain protein (NfL), phosphorylated tau-181 (pTau181), and amyloid beta (Aβ40, Aβ42), were measured via Simoa. A subset of participants (n = 7; age = 27.5 y/o [5.7]) and six controls (age = 25.1 y/o [4.5]) underwent PET scans to quantify brain amyloid and tau densities in AD sensitive brain regions. Y-DM adolescents exhibited lower plasma levels of Aβ40, Aβ42, and GFAP, and higher pTau181 compared to controls (p < 0.05), a pattern persisting into adulthood (p < 0.001). All biomarkers showed significant increases from adolescence to adulthood in Y-DM (p < 0.01), though no significant differences in brain amyloid or tau were noted between Y-DM and controls in adulthood. Preliminary evidence suggests that preclinical AD neuropathology is present in young people with Y-DM, indicating a potential increased risk of neurodegenerative diseases.

Alzheimer’s disease (AD) is the fifth leading cause of death among adults aged 65 and older, yet the onset and progression of the disease is poorly understood. What is known is that the presence of amyloid, particularly polymerized Aβ42, defines when people are on the AD continuum. Interestingly, as AD progresses, less Aβ42 is detectable in the plasma, a phenomenon thought to result from Aβ becoming more aggregated in the brain and less Aβ42 and Aβ40 being transported from the brain to the plasma via the CSF. We propose that extracellular vesicles (EVs) play a role in this transport. EVs are found in bodily fluids such as blood, urine, and cerebrospinal fluid and carry diverse “cargos” of bioactive molecules (e.g., proteins, nucleic acids, lipids, metabolites) that dynamically reflect changes in the cells from which they are secreted. While Aβ42 and Aβ40 have been reported to be present in EVs, it is not known whether this interaction is specific for these peptides and thus whether amyloid-carrying EVs play a role in AD and/or serve as brain-specific biomarkers of the AD process. To determine if there is a specific interaction between Aβ and EVs, we used isothermal titration calorimetry (ITC) and discovered that Aβ42 and Aβ40 bind to EVs in a manner that is sequence specific, saturable, and endothermic. In addition, Aβ incubation with EVs overnight yielded larger amounts of bound Aβ peptide that was fibrillar in structure. These findings point to a specific amyloid–EV interaction, a potential role for EVs in the transport of amyloid from the brain to the blood, and a role for this amyloid pool in the AD process.

Background: By age 40, all people with Down syndrome (DS) develop Alzheimer’s disease (AD) brain neuropathology, including Aβ plaques and neurofibrillary tangles of phosphorylated microtubule associated protein tau (MAPT), and most go on to develop early‐onset dementia. People with Down syndrome (DS) have triplication of chromosome 21 (trisomy 21) and they develop AD neuropathology due to triplication of the amyloid precursor protein gene (APP) which resides on chromosome 21. Here, we developed and characterized human induced pluripotent stem cell (hiPSC)‐derived neurons, astrocytes, microglia, and cerebral organoid (CO) models of DS and familial AD and used them to evaluate the AD‐related pathological mechanisms caused by trisomy 21.Method: We obtained hiPSC lines derived from individuals with DS, healthy control individuals (HC), and individuals with familial AD, including duplication of APP on one chromosome (APPdup) or carrying the London mutation in APP (APPV717I). All donors had the apolipoprotein E (APOE) ε3/ε3 genotype. We generated hiPSC‐derived neurons, astrocytes, microglia, and COs using validated techniques. We evaluated the hiPSC‐derived neural cells and COs at different ages for AD‐related neuropathology, neuroinflammation, and neurodegeneration by performing RNA sequencing, immunohistochemistry, immunoblotting, and enzyme‐linked immunosorbent assay.Result: AD‐related size phenotypes characteristic of neurodegeneration were observed in DS hiPSC‐based models relative to HC models. Dysregulated gene and protein expression of APP, MAPT, and APOE, in addition to chromosome 21 genes, were also observed in DS hiPSC‐based models. In particular, neuroinflammatory markers, especially those previously shown in APOE‐related pathways, were also increased in DS models.Conclusion: We characterized new hiPSC‐based models of DS and familial AD which will be useful for identifying commonalities in disease mechanisms as well as for dissecting the roles of chromosome 21 genes other than APP. We identified several AD‐related phenotypes in hiPSC‐based DS related to neuropathology, neuroinflammation, and neurodegeneration. We expect that these hiPSC‐based models will be useful for testing therapeutics in the future to address AD in people with DS, for which there is no cure.

Background: People with Type II diabetes mellitus (T2D) are at higher risk for developing Alzheimer’s disease (AD). T2D is characterized histopathologically by accumulation of pancreatic amyloid deposits comprised primarily of self‐aggregating amylin/human islet amyloid polypeptide (hIAPP). Amylin is implicated in both T2D and AD pathogenesis, and in T2D patients, pancreatic amyloid deposition occurs before and induces islet β‐cell apoptosis.Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) has demonstrated anti‐apoptotic, neuroprotective, neurotrophic, immunomodulatory, and regenerative effects in several neuronal injury models. We discovered that GM‐CSF treatment rapidly reduced cerebral amyloidosis and reversed cognitive impairment in transgenic AD models (Boyd et al, 2010), which led to the initiation and successful completion of a Phase 2 clinical trial (NCT01409915) using sargramostim (rhuGM‐CSF) in mild‐to‐moderate AD participants (Potter et al., 2021). Thus, we hypothesized the GM‐CSF might also reduce amyloid, prevent pancreatic apoptosis, and thereby reduce blood glucose in the hIAPP mouse model of T2D.Method: We investigated GM‐CSF treatment effects in hIAPP mice (Jackson strain Tg(Ins2‐IAPP)RHFSoel), which accumulate amylin amyloid in pancreatic islets. Hemizygous mice received daily subcutaneous injections (5 days/week for 23 total injections) of either recombinant murine GM‐CSF (5 µg/day or ∼167 µg/kg) or sterile saline, and were then euthanized, perfused according to CU Anschutz OLAR regulations, and pancreatic tissues obtained and processed for histopathology. Contrastingly, homozygous mice received 15 total injections and were monitored for 45 days post‐treatment, before euthanizing and perfusion procedures. In homozygous mice, blood glucose levels were measured 3 days/week (weeks 2‐4) and at 45 days post‐treatment. Unpaired t‐tests were used to determine significant differences (p<0.05) between treatment groups.Result: In pancreas of male GM‐CSF‐treated hemizygous mice (n = 9), Thioflavin‐S staining revealed significantly less pancreatic amyloid (p = 0.0140) and Caspase‐3 immunohistochemistry showed significantly less apoptosis (p = 0.0057), compared to the saline‐treated group (n = 7). In homozygous mice, GM‐CSF‐treated male (n = 5) and female (n = 3) mice showed significantly reduced blood glucose from baseline levels, which treatment effects were sustained 45 days post‐treatment (p<0.001 female, p = 0.0002 combined M/F), whereas saline treatments (n = 5 male; n = 3 female) had no effect.Conclusion: These preliminary data suggest therapeutic potential for GM‐CSF to prevent or treat T2D, and thereby reduce AD risk.