Summary of research evaluating GM6 Mechanisms of Action and Efficacy in Alzheimer’s Disease

Executive Summary

Alzheimer’s Disease (AD) is a common cause of dementia in the elderly and is characterized by prominent degenerative changes within the hippocampus. Development of drugs to treat or prevent AD is ongoing but thus far few treatments have been sufficiently promising to gain widespread use (e.g., donepezil, galantamine, rivastigmine and tacrine). GM6 is a peptide drug consisting of 6 amino acids (Phe-Ser-Arg-Tyr-Ala-Arg) developed by Genervon Biopharmaceuticals (Pasadena, CA). Through 20 years of preclinical and clinical research, Genervon has characterized pro-survival and neurotrophic effects of GM6 within the central and peripheral nervous systems. This document summarizes mechanisms of action for GM6 obtained through this work and provides evidence supporting in vivo evidence of efficacy for GM6 as a candidate AD therapy. GM6 interacts with multiple extracellular receptors and acts to antagonize melanocortin 5 receptor (MC5R), neuropeptide FF receptor 1 (NPFFR1), opioid related nociceptin receptor 1 (OPRL1) and domatostatin receptor 2 (SSTR2). GM6 further acts as an agonist of insulin and Notch receptors, while up-regulating expression of nerve growth factor receptor (NGFR) and fibroblast growth factor receptor like 1 (FGFRL1). Stimulation of these receptors modulates signal transduction pathways such as the insulin signaling and mitogen-activated protein kinase (MAPK) cascades, with involvement of key downstream proteins such as Shc, PTB domain, IRS-1, PI3 kinase, SOCS and Notch Intracellular domain (NICD). These signals converge to control the activity of C2H2 zinc finger transcription factors interacting with GC-rich DNA motifs, as well as helix-turn-helix homeodomain transcription factors interacting with AT-rich DNA motifs. Such transcription factors include hes family bHLH transcription factor 7 (HES7), GLI family zinc finger 1 (GLI1), homeobox D11 (HOXD11), and signal transducer and activator of transcription 3 (STAT3). By modulating activity of these and other transcription factors, GM6 controls the expression of AD-associated genes such as APOE, PLAU, NGFR, CACNA1G, CLU, RYR3, COX4I2, NDUFS2, NDUFB8, NDUFS4, COX10 and DOCK2, along with inflammation-associated genes involved in acute phase responses, lymphocyte proliferation, leukocyte migration, and cytokine production. Within the in vivo setting, these effects occur centrally and peripherally, as our studies demonstrate that GM6 penetrates the blood-brain barrier. Additional pharmacological studies demonstrated good penetrance into cells with an estimated brain:plasma ratio of 1.65. For in vivo validation, we studied the AD-like phenotype developing in APP/ΔPS1 double-transgenic mice. Effects of GM6 in this model were consistent with beta-secretase inhibition and alpha-secretase activation, favoring overall amyloid degradation and clearance. GM6 further decreased abundance of pro-inflammatory cytokines (TNF-alpha, IL-1beta, and TGF-beta) and increased abundance of nerve growth factor (NGF). Importantly, GM6 led to dose-dependent improvement in behavioral learning deficits in APP/ΔPS1 mice, as demonstrated by water maze testing performance. These results together demonstrate unique mechanisms of action mediating effects of GM6 on neuron survival, which we have now characterized with respect to extracellular receptors, signal transduction pathways, transcription factors and transcriptionally regulated genes. Moreover, our preclinical findings using the validated APP/ΔPS1 mouse model provide in vivo evidence for efficacy. These studies thus provide a strong rationale for phase II clinical studies of GM6 as a novel AD treatment based upon a novel and unique mechanism of action.

(1) Introduction

Alzheimer’s Disease (AD) is a common cause of dementia in the elderly and is characterized by widespread cortical atrophy affecting the frontal lobes along with prominent degenerative changes within the hippocampus. The clinical presentation of AD typically involves progressive decline of intellectual function over several years, including loss of recent memories (a leading indicator) with eventual decline of long-term memory and higher intellectual capacities (e.g., reading, speaking). Ultimately, disease progression may extend to motor dysfunction and even life-threatening paralysis. These features of AD overlap with some aspects of mild cognitive impairment due to normal aging, but often AD is clinically unmistakable and can be clearly distinguished from normal cognitive aging by its early age of onset and/or its rapid progression.

Macroscopic features of AD involve narrowing of gyri and widening of sulci leading to a characteristic ex vacuo ventriculomegaly. These gross anatomical changes are accompanied by the accumulation of extracellular senile beta-amyloid (Aβ) plaques in grey matter. The progression of AD has also been associated with neurofibrillary tangles consisting of intracellular hyperphosphorylated tau protein with insoluble cytoskeletal elements. The relative importance of extracellular Aβ plaques versus intracellular tau protein has been the subject of frequent debate as competing mechanisms accounting for AD onset and progression. It is possible that both Aβ and tau contribute to disease progression, with good evidence demonstrating that the abundance of tau tangles correlates with the severity of dementia and strong indication that Aβ is at least able to promote angiopathy that can predispose elderly patients to intracranial bleeding.

The exact etiology of AD therefore remains unclear, but ongoing research has identified genes and proteins associated with AD that are likely involved in the disease process. These include an amyloid precursor protein (APP) representing an immature form of beta-amyloid plaques that is encoded by a chromosome 21 locus. The presence of APP on chromosome 21 likely accounts for the increased frequency of AD in patients with Down syndrome (trisomy-21). AD has additionally been associated with certain protein variants, with risk of sporadic disease increased by ApoE4 and decreased by ApoE2. Presenilin-1 (PSEN1) and presenilin-2 (PSEN2) have likewise been closely associated with AD and may be especially important in the pathogenesis of familial AD characterized by an early age of onset.

The development of drugs to treat or prevent AD is ongoing but thus far only few treatments have been sufficiently promising to gain widespread use. The most frequently used agents have included indirect cholinergic agonists (i.e., AChE inhibitors or anticholinesterases) such as donepezil, galantamine, rivastigmine and tacrine. These drugs prevent central nervous system degradation of acetylcholine (ACh), which is a neurotransmitter largely produced within the basal nucleus of Meynert that is believed to play an important role in learning and memory. Memantine is an N-methyl-D-aspartate (NMDA) receptor antagonist has alternatively been used to treat AD patients and acts to prevent excessive and potentially pathological neuronal excitation mediated by glutamate and calcium. At present, AChE inhibitors and memantine are the only widely used drugs for AD treatment and no other pharmacological options have been approved since 2003. While these drugs may modify disease progression to a small degree, most patients will receive only symptomatic relief with either AChE inhibitors or memantine.

GM6 is a peptide drug consisting of 6 amino acids (Phe-Ser-Arg-Tyr-Ala-Arg) that has been developed by Genervon Biopharmaceuticals (Pasadena, CA). The drug is modeled upon an endogenous developmental-stage neurotrophic factor identified from the developing human nervous system (previously identified as MNTF1). Through 20 years of preclinical and clinical research, Genervon has characterized pro-survival and neurotrophic effects of GM6 within the central and peripheral nervous systems. This work has utilized a diverse range of model systems, including human studies, and has laid the groundwork for understanding cellular mechanisms by which GM6 may be effective as an AD treatment.

The purpose of this document is to summarize mechanisms of action for GM6 as an AD therapy, starting at the level of extracellular receptors engaged by GM6, with further characterization of signal transduction mechanisms linked to transcriptional responses. These downstream transcriptional responses have now been comprehensively characterized using genome-wide profiling technologies, and we have demonstrated that this response involves modulation of genes linked to key disease-associated processes, such as APOE, PLAU, NGFR, CACNA1G, CLU, RYR3, COX4I2, NDUFS2, NDUFB8, NDUFS4, COX10 and DOCK2. Finally, this report describes how we have extended our foundational preclinical investigations to vertebrate systems, including studies of a validated AD mouse model (APP/ΔPS1 mice transgenic mice). The totality of these studies have demonstrated that GM6 has unique effects compared to existing AD treatments, and have provided a “big picture” view of core mechanisms by which GM6 appears to act upon diverse cell types to influence disease progression.

(2) Receptors

Extracellular and intracellular receptors represent interaction points between a drug and individual target cells. These upstream pathway elements are the essential gatekeepers that ultimately control and initiate the cascade of biochemical effects leading to drug responses at the cellular level. GM6 was not designed to interact exclusively as an agonist or antagonist of any one receptor, but is instead hypothesized to act upon multiple receptors. We used enzyme fragment complementation (EFC) with β‐galactosidase assays to screen GPCR receptor libraries and have shown that GM6 has antagonist activity with respect to melanocortin 5 receptor (MC5R), neuropeptide FF receptor 1 (NPFFR1), opioid related nociceptin receptor 1 (OPRL1) and domatostatin receptor 2 (SSTR2). GM6 may also weakly antagonize cholinergic receptor muscarinic 4 (CHRM4), although this effect may be counterbalanced by up-regulation of CHRM4 following GM6 stimulation, which we have demonstrated in SH-SY5Y neuroblastoma cells. Using the same assays, we identified potential agonist activity with respect to adhesion G protein-coupled receptor B3 (ADGRB3/BAI3) and atypical chemokine receptor 3 (ACKR3/CXCR7).

The most post potent agonist activity of GM6 relates to insulin receptor alpha, for which GM6 acts as a positive allosteric modulator. The protein abundance of insulin receptor is also increased in GM6-stimulated cells, which we have confirmed through studies of multiple cell types, including human brain microvascular endothelial cells (HBMVECs), Ntera2 differentiated cells and GABAergic Neurons. We have identified additional receptors transcriptionally up-regulated by GM6 in SH-SY5Y neuroblastoma cells, including nerve growth factor receptor (NGFR) and fibroblast growth factor receptor like 1 (FGFRL1). GM6 further down-regulates the expression of key receptors mediating the inflammation response, such as TNF receptor superfamily member 19 (TNFRSF19), which may contribute to anti-inflammatory effects of the drug (discussed below). Finally, our gene expression profiling data have generated strong signals implicating the Notch and hedgehog signaling pathways, suggesting agonist activity with respect to Notch receptors (NOTCH1, 2, 3 and 4), patched 1 (PTCH1) and/or smoothened frizzled class receptor (SMO).

Collectively, these data have supported the hypothesis that, in contrast to many novel drug formulations, GM6 does not selectively interact with a single receptor target, but rather appears to interact with multiple receptors linked to a diverse set of pathways. These ligand-receptor interactions are further amplified by downstream transcriptional responses to GM6, which alter the abundance of additional receptors associated with neurogenesis and pro-survival responses.

(3) Signal Transduction

Signal transduction is the process by which extracellular signals are propagated intracellularly to activate signaling pathways that are in turn linked to downstream transcriptional responses. Our work has implicated a broad set of receptors mediating GM6 responses, and accordingly, we have identified a complex set of signaling pathways activated by GM6, which converge downstream to activate or inhibit core transcription factors.

Consistent with data demonstrating positive regulation of insulin receptor (IR), GM6-stimulated cells have increased phosphorylation of IR at tyrosine 972 in HBMVECs. This event likely indicates modulation of the insulin signaling cascade mediated by downstream proteins, such as Shc, PTB domain, IRS-1, PI3 kinase and SOCS. We have additionally observed decreased phosphorylation of the p85 regulatory subunit of phosphatidylinositide 3-kinase in Ntera2 differentiated cells. We expect that these signaling events would have downstream metabolic effects related to glucose uptake/release and the synthesis of carbohydrates, lipids and proteins, all of which may support neurogenesis and a broader pro-survival response. Other key proteins mediating GM6-directed signal transduction are likely to include Notch Intracellular domain (NICD), Deltex-1 (DTX1) and the mitogen-activated protein kinase (MAPK) cascade.

These findings have thus uncovered a diverse set of mediators linking extracellular signals to downstream cellular responses. These mediators transmit upstream signals and distribute effects of GM6 across a multi-pronged network of pathways, likely amplifying downstream responses and broadening the scope of biological processes involved in such responses. Our work has begun to characterize this chain of protein-protein interactions and phosphorylation events playing an essential role in cellular responses to GM6.

(4) Transcription Factors

Transcription factors are key convergence points for extracellular signaling pathways and play a critical role in coordinating a cell’s response to drugs or other stimuli. RNA-seq expression profiling in SH-SY5Y neuroblastoma cells has demonstrated that GM6 up-regulates genes are associated with C2H2 zinc finger transcription factors interacting with GC-rich motifs. In contrast, GM6 down-regulates genes associated with helix-turn-helix homeodomain transcription factors interacting with AT-rich motifs. Specific transcription factors up-regulated by GM6 include hes family bHLH transcription factor 7 (HES7), GLI family zinc finger 1 (GLI1), homeobox D11 (HOXD11), and signal transducer and activator of transcription 3 (STAT3). Up-regulation of HES7 and GLI1 expression by GM6 is consistent with Notch and hedgehog pathway activation, respectively. Moreover, DNA motifs known to interact with the HOXD11 and STAT3 transcription factors were enriched in sequence regions upstream of genes transcriptionally regulated by GM6. These findings have demonstrated that the gene expression response to GM6 is regulated by a core set of transcription factors. By applying genome-scale analyses such as RNA-seq we have begun to uncover the identity of transcription factors mediating responses to GM6, as well as the cis-regulatory codes linking these factors to specific DNA sequences.

(5) Transcriptional response

Using large-scale RNA-seq transcriptome analyses, we have identified 2867 protein-coding genes with expression significantly altered by GM6 (FDR < 0.10) in SH-SY5Y cells. A number of GM6-regulated genes we identified have an important influence on AD development and progression, with roles in genetic risk modulation, inhibition of Aβ production with augmentation of Aβ degradation/clearance, inhibition of intrinsic apoptosis cascades, and inhibition of neuroinflammation.

The mRNA encoding apolipoprotein E (APOE) was elevated nearly 3-fold following 48 hours of GM6 treatment (FC = 2.86; P = 0.000697). Variants of the APOE gene are the most significant genetic risk factor for late-onset AD. The gene encodes a chylomicron apoprotein mediating catabolism of lipoprotein constituents. The APOE e4 allele in particular has been identified as the pathological variant in AD, although it remains unclear whether the e4 variant augments AD risk through a gain of toxic function or a loss of protective function. Potentially, up-regulation of APOE expression by GM6 may have favorable compensatory effects in APOE e4 carriers to modulate genetic risk.

The role of extracellular Aβ plaques in AD pathology has been the subject of extensive debate. In addition to their putative role as mediators of AD-related dementia, Aβ plaques are believed to promote angiopathy that can increase long-term risk of intracranial bleeding. Transcriptional responses to GM6 suggest several mechanisms by which GM6 may attenuate Aβ plaque burden, including up-regulation of PLAU (plasminogen activator, urokinase), NGFR (nerve growth factor receptor), and CACNA1G (calcium voltage-gated channel subunit alpha1 G) as well as down-regulation of CLU (clusterin) and RYR3 (ryanodine receptor 3). PLAU encodes a secreted serine protease believed to contribute to Aβ plaques, whereas NGFR has protective functions against Aβ toxicity. CACNA1G encodes a voltage-sensitive calcium channel that contributes to calcium signaling involved in neurotransmitter release and AD appears to exacerbate age-related declines in CACNA1G expression. Clusterin (CLU) has emerged as a therapeutic target in AD as well as other diseases and its abundance is elevated in the AD brain and believed to influence Aβ plaque abundance. Finally, RYR3 encodes a ryanodine receptor that regulates intracellular calcium release, which may mediate Aβ plaque production and have acute pathological effects in later stages of AD. The totality of these gene expression responses to GM6 may favor reduced accumulation of Aβ plaques during the course of cognitive aging.

Elevated rates of apoptosis appear to be characteristic of AD and may contribute to neuronal loss. Such apoptotic neuronal death is mediated largely by cascades localized to mitochondria, and mitochondria additionally appear to facilitate Aβ plaque toxicity. Our transcriptional analyses have shown significant down-regulation of COX4I2 (cytochrome c oxidase subunit 4I2) following 48 hours of GM6 treatment (FC = 0.48; P = 7.71e-07). This gene encodes a subunit for the enzyme cytochrome c oxidase (COX), which is the terminal enzyme of the mitochondrial respiratory chain. Other mitochondria-associated genes down-regulated by GM6 include NDUFS2, NDUFB8, NDUFS4 and COX10. These results suggest that GM6 may have inhibitory effects on mitochondrial activity and/or abundance, which could contribute to decreased apoptosis through the intrinsic pathway and/or impaired production of reactive oxygen species.

Chronic neuroinflammation contributes to AD and multiple other neurodegenerative diseases of aging. Regulators of neuroinflammation have therefore emerged as therapeutic targets for such diseases. As a component of inflammatory reactions, DOCK2 (dedicator of cytokinesis 2) has been demonstrated to facilitate lymphocyte migration and is highly expressed in peripheral blood leukocytes as well as microglia. We have shown that DOCK2 expression is down-regulated as an early (6 hours) response to GM6 treatment (FC = 0.38; P = 7.51e-06), although extended (24 hours) GM6 treatment leads to increased DOCK2 expression (FC = 2.09; P = 0.0168). The short-term response may be beneficial in the setting of AD, since DOCK2 regulates innate immune status of microglial cells within the AD brain, and ablation of DOCK2 was shown to reduce Aβ plaque levels suggesting that DOCK2 may be a valid therapeutic target.

To explore these anti-inflammatory responses further, we have extended our expression profiling analyses to include immune-associated cell types, such as jurkat T lymphocyte cells. In jurkat T cells, genes down-regulated by GM6 were associated with acute-phase response proteins, immune response, inflammation and production of cytokines such as IL-1 and TNF. Proteins involved in the acute phase response are known to exhibit changes in serum concentration during inflammation. These proteins, such as C reactive protein, can be used to monitor systemic inflammation and liver is often a site of synthesis. Genes down-regulated by GM6 were associated with the acute-phase response (FN1, SERPINA1, FGA, AHSG, SERPINA3, SERPINF2, ORM1, F2). Notably, FGA encodes a component of fibrinogen, which is a coagulation factor whose abundance may correlate with erythrocyte sedimentation rate (ESR).

Genes down-regulated by GM6 in jurkat cells were additionally associated with immune system processes (FGA, APOA1, FGB, AMBP, FABP1, GPC3, APOA2, EGR1, DHRS2, ORM1, SP2, CYP27B1, NOTCH1, ZFAT), humoral immune response (FGA, FGB, NOTCH1) and regulation of inflammatory response (AHSG, A2M, C3, AGT). Decreased genes were also involved in triggering of immune responses and other downstream steps of immune reactions, such as response to lipopolysaccharide (APOB, NR1D1, CYP27B1, NOTCH1), response to bacterium (APOB, LYZ, FGA, FGB, PGC, NR1D1, CYP27B1, NOTCH1), regulation of immune effector process (RBP4, APOA1, FABP1, APOA2, PGC), positive regulation of lymphocyte proliferation (IGF2, JAK3, IRS2) and leukocyte migration (FN1, APOB, CD84, DBH, F2).

Cytokines are important mediators of immune and inflammatory reactions. Genes down-regulated by GM6 in the jurkat cell line were additionally linked to regulation of cytokine biosynthetic process (APOA2, TNFRSF8, THBS1), regulation of cytokine production (C3, AGT, SERPINF2, ORM1, CREBBP), response to interleukin-1 (FGB, EGR1, CREBBP), regulation of TNF production (TNFRSF8, ORM1, THBS1) and cellular response to TNF (APOB, APOA1, KRT8, KRT18). These findings demonstrate multiple gene expression responses in an immune-associated cell type (jurkat T lymphocytes) that may correspond to anti-inflammatory effects in vivo. 

(6) Pharmacology

Since AD is thought to involve the death of hippocampal and other neurons within the central nervous system, it is essential for any candidate AD therapy to be able to cross the blood-brain barrier. We have demonstrated that GM6 penetrates the blood brain barrier and is well absorbed by neuronal tissues. In our studies of C57BL/6 mice, endogenous GM6 levels were estimated to be 0.4 μM in the brain, but following GM6 injection (0.2 mg/kg), GM6 brain levels increased to 1.760 μM (4.4-fold increase). With more concentrated GM6 injection (2 mg/kg), brain levels increased to 12.92 μM (32.3-fold increase). These studies have confirmed the presence of an endogenous GM6 protein subunit within the central nervous system, and have additionally demonstrated that GM6 is able to cross the blood brain barrier to reach upper motor neurons within the central nervous system. Additional pharmacological studies demonstrated that GM6 has strong drug-like properties and good penetrance into tissues with estimated volume of distribution of 7.8 L/kg. GM6 additionally has good penetrance into cells with a cell partition of 5.2, indicating an intracellular GM6 concentration 5.2 times greater than its extracellular concentration. The estimated brain:plasma ratio is 1.65 consistent with good penetration into neuronal tissues. The tissue half-life was estimated to be 5.8 hours using microsomal clearance assays, and we have demonstrated that this half-life is not influenced by the presence of other pharmacological agents commonly used to treat neurodegenerative diseases.

(7) Mouse model of AD

Findings outlined above suggested that GM6 has in vitro activities consistent with a neurotrophic factor, with pharmacological properties that would allow stable in vivo delivery of GM6 to cells following intravenous administration. To further evaluate in vivo effects of GM6, therefore, we utilized the AD-like phenotype developing in APP/ΔPS1 double-transgenic mice, which express a familial AD (FAD) mutant AβPP and mutant presenilin 1 (PS1) protein (DeltaE9). The combined effects of these mutant proteins leads to development of a highly penetrant and well-validated AD-like phenotype that has often been utilized in preclinical AD studies. In our studies, we evaluated APP/ΔPS1 double-transgenic mice with a hybrid C3H/HeJ*C57BL/6J genetic background. Our study design included mice receiving vehicle or daily GM6 injections at one of two doses (1 or 5 mg/kg per day; n = 20 mice per group). Treatments were given over a 4 month period starting in young mice at 3 months of age.

The study allowed us to evaluate in vivo effects of GM6 on brain histology, biochemical assays, neuroinflammation and behavioral manifestations in the validated APP/ΔPS1 AD-like phenotype. Histological effects of GM6 in APP/ΔPS1 double-transgenic mice were consistent with reduced disease burden, with decreased numbers of amyloid plaques and Aβ peptide (Aβ1-40 and Aβ1-42). Biochemical assays indicated that GM6 altered amyloid metabolism in two key respects. First, GM6 decreased abundance of the β-secretase cleavage product CTFβ, consistent with inhibition of the amyloidogenic pathway beta-secretase. Secondly, GM6-treated mice exhibited increased brain levels of sAPPα, suggesting activation of the non-amyloidogenic pathway component alpha-secretase. These two effects, beta-secretase inhibition and alpha-secretase activation, suggest that GM6 may replicate the activity of compounds targeting amyloid metabolism, several of which have recently been evaluated as AD treatments (e.g., verubecestat, lanabecestat and LY2886721).

Biochemical analysis of brain tissues from GM6-treated mice further identified neurotrophic and anti-inflammatory mechanisms by which GM6 may combat AD progression. GM6 increased abundance of nerve growth factor (NGF), which in previous studies has been shown to activate neurons in brains from AD patients and induce axonal sprouting. Biochemical assays further demonstrated suppression of neuroinflammation in APP/ΔPS1 double-transgenic mice, based upon reduced numbers of CD68-positive microglial cells and glial fibrillary acidic protein (GFAP)-positive astrocytes. One possible mechanism mediating this effect appeared to be decreased abundance of cathepsin B, which is a pro-inflammatory protein also targeted by some AD drugs. Additionally, GM6 significantly decreased brain abundance of key pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and TGF-beta.

Our analyses of APP/ΔPS1 double-transgenic mice identified unique effects of GM6 related to amyloid metabolism and neurotrophic or anti-inflammatory mechanisms. Ultimately, however, the most important aspect of GM6 was its effects on the behavioral learning deficits observed in this AD mouse model. To measure spatial learning ability, we utilized Morris water maze testing and measured the amount of time required by mice to locate an escape platform in the presence of learned visual cues located outside of the swimming pool. For GM6-treated mice, the average time required was 27.6 seconds, whereas nearly twice this amount of time was needed on average (50.2 seconds) in vehicle-treated mice. As a measure of memory retention, we next evaluated the ability of mice to swim to the appropriate area after the escape platform had been removed. GM6-treated mice spent 3-4X as long in the correct escape platform region as compared to the vehicle treated mice (1 mg/kg GM6: 35.8 seconds; 5 mg/kg GM6: 40.3 seconds; vehicle: 10.0 seconds). These functional data corroborate our biochemical and histological analyses and suggest that, through neurotrophic, anti-inflammatory, and amyloid catabolism effects, GM6 improves the AD-like phenotype that develops in APP/ΔPS1 transgenic mice.

(8) Conclusion

AD is a challenging disease that lacks highly effective pharmacological treatment options. Through decades of preclinical and clinical research, we developed the peptide drug GM6 as a potential treatment to improve neuron survival. GM6 is a small peptide drug consisting of only 6 amino acids that is small enough to cross the blood-brain barrier and act directly upon the central nervous system. The safety of GM6 has been demonstrated in phase 1 and 2 clinical studies. Findings outlined in this summary demonstrate our progress in characterizing unique mechanisms of action of GM6 as a centrally-acting AD treatment. These mechanisms involve multiple extracellular receptors and activation of intracellular signaling cascades, leading to altered transcription of hundreds of genes with broad hormone-like effects on cellular physiology. We have hypothesized that these effects can effectively promote neuron survival in the setting of AD, resulting in the inhibition of symptom progression. In support of this, we have demonstrated efficacy of GM6 for treatment of the AD-like phenotype in a validated mouse model (APP/ΔPS1 double-transgenic mice). Within this model, GM6 appears to hinder amyloid deposition through beta-secretase inhibition and alpha-secretase activation, down-regulation of pro-inflammatory cytokines (TNF-alpha, IL-1beta, and TGF-beta), and increased nerve growth factor (NGF) abundance. We have further shown that cognitive decline, as evaluated by water maze performance, is prevented in GM6-treated APP/ΔPS1 mice. These results together demonstrate unique mechanisms of action mediating effects of GM6 on neuron survival, which we have now characterized at the level of extracellular receptors, signal transduction pathways, transcription factors and transcriptionally regulated genes. Our findings provide a strong rationale for human phase 1/2 studies of GM6 as an AD treatment based upon a novel mechanism of action distinct from existing AD drug candidates.