humanin and Alzheimer-Disease

Humanin (HN) is an endogenous peptide factor and known as a member of mitochondrial-derived peptides. We first found the gene encoding this novel 24-residue peptide in a brain of an Alzheimer's disease (AD) patient as an antagonizing factor against neuronal cell death induced by AD-associated insults.. This review presents an overview of HN actions in AD-related conditions among its wide range of action spectrum as well as a brief history of the discovery.. HN exhibits multiple intracellular and extracellular anti-cell death actions and antagonizes various AD-associated pathomechanisms including amyloid plaque accumulation.. This review concisely reflects accumulated knowledge on HN since the discovery focusing on its functions related to AD pathogenesis and provides a perspective to its potential contribution in AD treatments.

Topics: Alzheimer Disease; Apoptosis; Brain; Cell Death; Humans; Intracellular Signaling Peptides and Proteins; Peptides

Alzheimer's disease (AD), Parkinson's disease (PD), and age-related macular degeneration (AMD) are common among neurodegenerative diseases, but investigations into novel therapeutic approaches are currently limited. Humanin (HN) is a mitochondrial-derived peptide found in brain tissues of patients with familial AD and has been increasingly investigated in AD and other neurodegenerative diseases.. In this review, we summarize and discuss the effects of HN on the pathology of neurodegenerative diseases and cognition based on several studies from preclinical to clinical models. The association between cardiac ischemia-reperfusion (I/R) injury and brain are also included. Findings from in vitro studies and those involving mice provide the most fundamental information on the impact of HN and its potential association with clinical studies.. HN plays a considerable role in countering the progression and neuropathology of AD. Inhibition and reduction of oxidative stress and neuroinflammation of the original amyloid hypothesis is the mainstay mechanism. Multiple intracellular mechanisms will be elucidated, including those involved in the anti-apoptotic signaling cascades, the insulin signaling pathway, and mitochondrial function, and especially autophagic activity. These beneficial roles are also found following cardiac I/R injury. Cognitive improvement was found to be related to maintenance of synaptic integrity and neurotransmitter modulation. Small humanin-like peptide 2 demonstrates the neuroprotective effects in PD and AMD via prevention of mitochondrial loss.. Comprehensive knowledge of HN effects on cognition and neurodegenerative diseases emphasizes its potential to treat a viable disease, as it ameliorates the pathogenesis of the disease.

Topics: Alzheimer Disease; Animals; Apoptosis Regulatory Proteins; Cognition; Humans; Intracellular Signaling Peptides and Proteins; Macular Degeneration; Mice; Neurodegenerative Diseases; Parkinson Disease; Peptides; Reperfusion Injury

Humanin is a 24-amino acid, secreted bioactive peptide that prevents various types of cell death and improves some types of cell dysfunction. Humanin inhibits neuronal cell death that is caused by a familial Alzheimer's disease (AD)-linked gene via binding to the heterotrimeric Humanin receptor (htHNR). This suggests that Humanin may play a protective role in AD-related pathogenesis. Calmodulin-like skin protein (CLSP) has recently been identified as a physiological agonist of htHNR with 10(5)-fold more potent anti-cell death activity than Humanin. Humanin has also shown to have protective effects against some metabolic disorders. In this review, the broad range of functions of Humanin and the functions of CLSP that have been characterized thus far are summarized.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Calcium-Binding Proteins; Cell Death; Humans; Intracellular Signaling Peptides and Proteins; Neurons

The prevalence of Alzheimer's disease (AD) is higher among type 2 diabetes mellitus (T2DM) patients. In T2DM patients, the progression of AD is more rapid. Furthermore, several pathophysiological pathways are common to AD and T2DM. Humanin is a recently introduced, mitochondrial-derived peptide with neuroprotective effects. Humanin can alter the mechanisms involved in AD and T2DM pathogenesis. Insulin resistance as well as oxidative stress has been shown to be associated with increased amyloid deposition in brain neurons and islet beta cells. Moreover, advanced glycation end products and lipid metabolism disorders are common pathways of oxidative stress and low-grade systemic inflammation in AD and T2DM. These common pathways may explain AD and T2DM pathogenesis and suggest common treatments for both diseases. Treatments for T2DM and AD attempt to slow cognitive decline, and recent investigations have focused on agents that may alter pathways common to AD and T2DM pathogenesis. Non-steroidal antiinflammatory drugs, such as interleukin-1 antagonists and statins, are possible drug candidates for both AD and T2DM.

Topics: Alzheimer Disease; Apolipoproteins E; Diabetes Mellitus, Type 2; Humans; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Oxidative Stress

Despite a bulk of evidence supporting the idea that increased neurotoxic insults lead to Alzheimer's disease (AD), the possibility still remains that insufficiency of an endogenous defense system contributes to the disease progression. Humanin is a bioactive peptide that is likely to inhibit both neuronal death and dysfunction only related to AD by binding to a Humanin receptor on the cell-surface and by activating a STAT3-mediated signal, preventing the onset of dementia. A couple of recent studies presented evidence suggesting that the Humanin signal is decreased in neurons of AD patients. If this is the case, the restoration or activation of the Humanin signal in neurons may change the course of AD.

Topics: Alzheimer Disease; Animals; Cell Death; Humans; Intracellular Signaling Peptides and Proteins; Models, Biological; Nerve Degeneration; Signal Transduction

Humanin (HN), a short amino acid peptide, protects neurons as well as other cells from amyloid β-induced toxicities and other stresses. A number of HN binding proteins have been identified and their involvements in HN-mediated neuroprotection have been suggested in some cases. However, the way HN binds to the target molecules has never been clarified. Here we will review the structures of HN and HN analogs in solution as a function of solvent conditions and attempt to relate their structural characteristics to the functional properties.

Topics: Alzheimer Disease; Humans; Intracellular Signaling Peptides and Proteins; Neuropeptides; Neuroprotective Agents

Alzheimer's disease (AD) is a prevalent dementia-causing neurodegenerative disease. Neuronal death is closely linked to the progression of AD-associated dementia. Accumulating evidence has established that a 24-amino-acid bioactive peptide, Humanin, protects neurons from AD-related neuronal death. A series of studies using various murine AD models including familial AD gene-expressing transgenic mice have shown that Humanin is effective against AD-related neuronal dysfunction in vivo. Most recently, it has been shown that Humanin inhibits neuronal cell death and dysfunction by binding to a novel IL-6-receptor-related receptor(s) on the cell surface involving CNTFRalpha, WSX-1, and gp130. These findings suggest that endogenous Humanin [or a Humanin-like substance(s)] may suppress the onset of AD-related dementia by inhibiting both AD-related neuronal cell death and dysfunction.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Cell Death; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Nerve Tissue Proteins; Neurons

Topics: Alzheimer Disease; Animals; Drug Design; Humans; Intracellular Signaling Peptides and Proteins; Janus Kinase 2; Nerve Tissue Proteins; STAT3 Transcription Factor; Synaptic Transmission

Alzheimer's disease (AD) is the most prevalent neurological disease with dementia. AD-related dementia is caused by death and dysfunction of neurons involved in cognitive function. It has been generally believed that increased levels of toxic amyloid-betas (Abetas) are linked to the occurrence of neuronal death as well as dysfunction (Abeta cascade theory). Consequently, lowering levels of toxic Abetas in the brain is considered to be central for therapy of AD. Multiple drug candidates based on this therapeutic strategy have been developed and are being vigorously developed. Some clinical studies have indicated that this strategy is effective. In addition to this theory, Abeta-independent pathomechanisms have been shown to contribute to the progression of AD-related dementia, justifying alternative strategies for AD treatment that are effective against Abeta-independent pathomechanisms. A possible therapeutic strategy belonging to them is to directly suppress AD-related neuronal death and dysfunction. A series of studies indicated that a 24-amino-acid bioactive peptide named Humanin was shown to inhibit neuronal cell death induced by enforced expression of familial AD-related genes. Humanin also protected neurons from being killed by toxic Abetas in vitro. In addition, neuronal dysfunction-associated dementia of mice caused by muscarinic receptor antagonists and intracranially injected toxic Abetas was ameliorated by Humanin therapy. Multiple studies have indicated the existence of a putative specific Humanin receptor on the cell membrane. These results together suggest that an endogenous AD-related humoral factor(s) may inhibit the progression of AD-related dementia by inhibiting both neuronal cell death and dysfunction in vivo. Malfunction of this self-defense mechanism is also hypothesized to be another etiology or an aggravator of AD. Moreover, from a standpoint of AD therapy, stimulation of the AD defense mechanism by a potent Humanin derivative is a promising alternative strategy for AD treatment. The present patents cover Humanin and the methods of its clinical usage.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Cell Death; Humans; Intracellular Signaling Peptides and Proteins; Neurons

Alzheimer's disease (AD) is a complex disease, involving multiple factors such as the production of aggregation-prone amyloid beta (Abeta) peptides, the formation of fibrillarly tangles of microtubule-associating proteins, Tau, and the polymorphism of cholesterol binding protein, APOE4. While understanding the mechanism of AD and the involvement of key players should lead to rational drug discovery against this disease, a traditional screening approach should also work for identifying drugs using AD models. We have used a cellular AD model, in which a cell death was induced by AD-causing neurotoxicities, and then screened the genes, which rescued the cells from the cell death. This resulted in isolation of a gene encoding a novel 24-amino acid long peptide, termed Humanin (HN), which protected neuronal cells at approximately microM level. Surprisingly, these gene products and the synthetic peptides not only protected neurons from cell death induced by Abeta-related neurotoxicities, but also Abeta-unrelated neurotoxicities. While a broad range of activities of HN against AD-related insults is discovered, the detailed mechanism of its action is still obscure. Structure analysis of HN showed that it is largely disordered and flexible at low peptide concentrations and heavily aggregates at high concentrations. Interestingly, one of the HN analogs, which is 10000-times more active than the parent HN molecule (i.e. active below nM range), was found to be monomeric. Based on findings of structural analyses, we propose here that membrane environment may enable HN to achieve high affinity for target protein(s) with multiple-transmembrane domains, such as G-protein coupled receptors.

Topics: Alzheimer Disease; Animals; Binding Sites; Cell Death; Drug Design; Humans; Intracellular Signaling Peptides and Proteins; Ligands; Neurons; Neuroprotective Agents; Protein Conformation; Structure-Activity Relationship

Humanin (HN), a 24-amino-acid neuroprotective peptide, was originally found in the occipital lobe of an autopsied Alzheimer's disease (AD) patient. HN inhibits neuronal death by binding to its specific receptor on the cell membrane and triggering a Jak2/STAT3 prosurvival pathway. The activation of this pathway may represent a therapeutic approach to AD. HN also exhibits neuroprotective activity against toxicity by familial amyotrophic lateral sclerosis (ALS)-related mutant superoxide dismutase (SOD1). Recent investigations established that AGA-(C8R)-HNG17, a 17-amno-acid derivative of HN, is 10(5) times more potent as a neuroprotective than HN; at 10-picomolar and higher concentrations in vitro it completely suppresses neuronal death. Moreover, a 26-amino-acid peptide colivelin (CL), composed of activity-dependent neurotrophic factor (ADNF) C-terminally fused to AGA-(C8R)-HNG17, provides complete neuroprotection at 100-femtomolar or higher concentrations in vitro. A series of experiments using mouse AD and ALS models further established the efficacy of HN derivatives, including CL, against these diseases in vivo. HN and CL can be viewed as drug candidates for neuronal death suppression therapy in AD or ALS.

Topics: Alzheimer Disease; Amyotrophic Lateral Sclerosis; Animals; Cell Death; Humans; Intracellular Signaling Peptides and Proteins; Models, Biological

Humanin (HN), a recently identified neuroprotective factor against Alzheimer's disease-related insults, has been reported to function as an anti cell-death factor through multiple mechanisms. One mechanism, revealed in a glioblastoma cell line, involves the apoptosis-inducing protein Bax. This, in addition to the fact that HN is produced in certain normal tissues, such as testis, implies a potential role of HN in oncogenesis. A second mechanism, in neuronal cells, is via a putative cell-surface receptor. It is through this mechanism that HN exhibits its neuroprotective activity.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Apoptosis; bcl-2-Associated X Protein; Brain Chemistry; Humans; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; Neuroprotective Agents; Peptides; Proteins; Proto-Oncogene Proteins c-bcl-2; Sequence Alignment

Neuronal cell death is the central abnormality occurring in brains suffering from Alzheimer's disease (AD). The notion that AD is a disease caused by loss of neurons points toward suppression of neuronal death as the most important therapeutic target. Nevertheless, the mechanisms for neuronal death in AD are still relatively unclear. Three known mutant genes cause familial AD (FAD): amyloid precursor protein, presenilin 1, and presenilin 2. Detailed analysis of cytotoxic mechanisms of the FAD-linked mutant genes reveals that they cause neuronal cell death at physiologically low expression levels. Unexpectedly, cytotoxic mechanisms vary depending on the type of mutations and genes, suggesting that various mechanisms for neuronal cell death are involved in AD patients. In support of this, activity-dependent neurotrophic factor, basic fibroblast growth factor, and insulin-like growth factor-I can completely protect neurons from beta-amyloid (A beta) cytotoxicity but exhibit incomplete or little effect on cytotoxicity by FAD mutant genes. By contrast, Humanin, a newly identified 24-residue peptide, suppresses neuronal cell death by various FAD mutants and A beta, whereas this factor has no effect on cytotoxicity from AD-irrelevant insults. Studies investigating death and survival of neuronal cells exposed to AD insults will open a new horizon in developing therapy aimed at neuroprotection.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Brain; Cell Death; Gene Expression; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mutation; Neurons; Presenilin-1; Proteins

The mechanism for neuronal cell death by familial Alzheimer's disease (FAD) genes turned out to consist of various elemental combinations by the different types of cytotoxicity. We therefore tried a new approach toward identifying novel anti-AD suppressors of neuronal death, termed disease-based death trap screening. The identified genes were classified into three categories. In the first category, there were known anti-cell death genes; in the second, there were known genes with unknown function; and in the third, there were novel genes. The cDNA that encodes a 24-residue peptide, termed HN, is in the third category. HN protects neuronal death caused not only by all known kinds of FAD genes (mutant APP, PS1 and PS2), but also by anti-APP antibody and Abeta peptides, but not by long polyQ or SOD1 mutants. HN is a secretory peptide and exerts its protective function from the outside of the cell. The function of HN strictly depends on the structure. C8A-HN lost the activity and S14G-HN had approximately 1000 times increased potency of its action. Immunoblot analysis detected 3-kDa HN immunoreactive peptide in the testis and the colon in 3-week-old mice and only in the testis in 12-week-old mice. Notably, no HN immunoreactivity was detected in the brain. However, in an AD brain, not in an age-matched control, HN immunoreactivity was detected in neurons in the occipital lobe and in reactive glias in the hippocampal sections. The specific binding for HN exists on the neuronal cells and the rescue action of HN is specifically inhibited by genistein but not by wortmannin, suggesting that HN acts through the neuronal surface receptor linked to certain tyrosine kinases, but different from typical receptor tyrosine kinases. This study will provide a new insight into the research of AD.

Topics: Alzheimer Disease; Animals; Brain; Cell Death; Cell Survival; Humans; Intracellular Signaling Peptides and Proteins; Mice; Neurons; Protein-Tyrosine Kinases; Proteins; Receptors, Cell Surface; Signal Transduction

Humanin (HN) is an endogenous 24-residue peptide that was first identified as a protective factor against neuronal death in Alzheimer's disease (AD). We previously demonstrated that the highly potent HN derivative HNG (HN with substitution of Gly for Ser14) ameliorated cognitive impairment in AD mouse models. Despite the accumulating evidence on the antagonizing effects of HN against cognitive deficits, the mechanisms behind these effects remain to be elucidated.. The extracellular fluid in the hippocampus of wild-type young mice was collected by microdialysis and the amounts of neurotransmitters were measured. The kinetic analysis of exocytosis was performed by amperometry using neuroendocrine cells.. The hippocampal acetylcholine (ACh) levels were increased by intraperitoneal injection of HNG. HNG did not affect the physical activities of the mice but modestly improved their object memory. In a neuronal cell model, rat pheochromocytoma PC12 cells, HNG enhanced ACh-induced dopamine release. HNG increased ACh-induced secretory events and vesicular quantal size in primary neuroendocrine cells.. These findings suggest that HN directly enhances regulated exocytosis in neurons, which can contribute to the improvement of cognitive functions.. The regulator of exocytosis is a novel physiological role of HN, which provides a molecular clue for HN's effects on brain functions under health and disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Apoptosis Regulatory Proteins; Intracellular Signaling Peptides and Proteins; Kinetics; Mice; Rats

Fibroblast Growth Factor 21 (FGF21), Growth Differentiation Factor 15 (GDF15), and Humanin (HN) are mitochondrial stress-related mitokines, whose role in health and disease is still debated. In this study, we confirmed that their plasma levels are positively correlated with age in healthy subjects. However, when looking at patients with type 2 diabetes (T2D) or Alzheimer's disease (AD), two age-related diseases sharing a mitochondrial impairment, we found that GDF15 is elevated in T2D but not in AD and represents a risk factor for T2D complications, while FGF21 and HN are lower in AD but not in T2D. Moreover, FGF21 reaches the highest levels in centenarian' offspring, a model of successful aging. As a whole, these data indicate that (i) the adaptive mitokine response observed in healthy aging is lost in age-related diseases, (ii) a common expression pattern of mitokines does not emerge in T2D and AD, suggesting an unpredicted complexity and disease-specificity, and (iii) FGF21 emerges as a candidate marker of healthy aging.

Topics: Aged, 80 and over; Alzheimer Disease; Diabetes Mellitus, Type 2; Fibroblast Growth Factors; Growth Differentiation Factor 15; Healthy Aging; Humans; Intracellular Signaling Peptides and Proteins

Humanin is a member of a new family of peptides that are encoded by short open reading frames within the mitochondrial genome. It is conserved in animals and is both neuroprotective and cytoprotective. Here we report that in

Topics: Adult; Aged, 80 and over; Alzheimer Disease; Animals; Animals, Genetically Modified; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Case-Control Studies; Child; Cohort Studies; DNA, Mitochondrial; Female; Forkhead Transcription Factors; Gene Dosage; Humans; Infant, Newborn; Intracellular Signaling Peptides and Proteins; Longevity; Macaca mulatta; MELAS Syndrome; Mice; Middle Aged; Mitochondria; Models, Animal; Mole Rats; Pregnancy; Young Adult

Humanin (HN), a 24-amino acid bioactive peptide, has been shown to increase cell survival of neurons after exposure to Aβ and NMDA-induced toxicity and thus could be beneficial in the treatment of Alzheimer's disease (AD). The neuroprotection by HN is reported to be primarily through its agonist binding properties to the gp130 receptor. However, the peptidic nature of HN presents challenges in its development as a therapeutic for AD. We report here for the first time the elucidation of the binding site of Humanin (HN) peptide to the gp130 receptor extracellular domain through modeling and the synthesis of small molecule mimetics that interact with the HN binding site on the gp130 receptor and provide protection against NMDA-induced neurotoxicity in primary hippocampal neurons. A brain permeable small molecule mimetic was identified through exploratory medicinal chemistry using microfluidic flow chemistry to facilitate the synthesis of new analogues for screening and SAR optimization.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Cell Death; Hippocampus; Intracellular Signaling Peptides and Proteins; N-Methylaspartate; Neurons

Humanin and calmodulin-like skin protein (CLSP) inhibits Alzheimer disease (AD)-related neuronal cell death via the heterotrimeric humanin receptor in vitro. It has been suggested that CLSP is a central agonist of the heterotrimeric humanin receptor in vivo. To investigate the role of CLSP in the AD pathogenesis in vivo, we generated mouse CLSP-1 transgenic mice, crossed them with the APPswe/PSEN1dE9 mice, a model mouse of AD, and examined the effect of CLSP over-expression on the pathological phenotype of the AD mouse model. We found that over-expression of the mouse CLSP-1 gene attenuated spatial learning impairment, the loss of a presynaptic marker synaptophysin, and the inactivation of STAT3 in the APPswe/PSEN1dE9 mice. On the other hand, CLSP over-expression did not affect levels of Aβ, soluble Aβ oligomers, or gliosis. These results suggest that the CLSP-mediated attenuation of memory impairment and synaptic loss occurs in an Aβ-independent manner. The results of this study may serve as a hint to the better understanding of the AD pathogenesis and the development of AD therapy.

Topics: Alzheimer Disease; Animals; Brain; Calpain; Cytoskeletal Proteins; Intracellular Signaling Peptides and Proteins; Learning Disabilities; Maze Learning; Mice; Mice, Transgenic; Nerve Tissue Proteins; Neuroprotection; Presenilin-1; STAT3 Transcription Factor; Synaptophysin

Humanin (HN) is an endogenous 24-residue peptide. A highly potent HN derivative, S14G-HN, which has a substitution of serine 14 to glycine, reduced amyloid burden and suppressed cognitive impairment in a mouse model of Alzheimer's disease. S14G-HN also suppressed amnesia induced by a muscarinic receptor antagonist in rodents. To understand the effects of HN on brain function, we tested the effect of S14G-HN on diazepam (DZP)-induced memory impairment and anxiety in mice using the object recognition test and zero-maze test, respectively. Intraperitoneal injection of S14G-HN reversed the DZP-induced memory deficit, whereas no significant change was observed in behavioral markers of anxiety. S14G-HN had no effect on locomotor activity in either test, indicating that S14G-HN did not affect physical functioning or motivation. These results suggest that HN preferentially influences cognitive function but not emotional function in the central nervous system.

Topics: Alzheimer Disease; Animals; Anticonvulsants; Cognition; Diazepam; Disease Models, Animal; Intracellular Signaling Peptides and Proteins; Male; Maze Learning; Memory Disorders; Mice; Neuroprotective Agents

Humanin is a small secreted peptide that is encoded in the mitochondrial genome. Humanin and its analogues have a protective role in multiple age-related diseases including type 2 diabetes and Alzheimer's disease, through cytoprotective and neuroprotective effects both in vitro and in vivo. However, the humanin-mediated signaling pathways are not well understood. In this paper, we demonstrate that humanin acts through the GP130/IL6ST receptor complex to activate AKT, ERK1/2, and STAT3 signaling pathways. Humanin treatment increases phosphorylation in AKT, ERK 1/2, and STAT3 where PI3K, MEK, and JAK are involved in the activation of those three signaling pathways, respectively. Furthermore, old mice, but not young mice, injected with humanin showed an increase in phosphorylation in AKT and ERK1/2 in the hippocampus. These findings uncover a key signaling pathway of humanin that is important for humanin's function and also demonstrates an age-specific in vivo effect in a region of the brain that is critical for memory formation in an age-dependent manner.

Topics: Age Factors; Alzheimer Disease; Animals; Cell Line, Tumor; Cytokine Receptor gp130; Diabetes Mellitus, Type 2; HEK293 Cells; Hippocampus; Humans; Intracellular Signaling Peptides and Proteins; Janus Kinases; Male; MAP Kinase Kinase Kinases; Memory; Mice; Mice, Inbred C57BL; Mitochondria; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT3 Transcription Factor

Humanin, a short bioactive peptide, inhibits a variety of cell deaths. Humanin-mediated inhibition of neuronal cell death, caused by an Alzheimer's disease (AD)-linked mutant gene occurs via binding of Humanin to its heterotrimeric Humanin receptor (htHNR), which results in the activation of the Janus-associated kinases (JAKs) and signal transducer and activator and transcription 3 (STAT3) signaling pathway. A previous study demonstrated that the Humanin-induced activation of the htHNR/JAK2/STAT3 signaling pathway leads to increased expression of SH3 domain-binding protein 5 (SH3BP5), which is an essential effector of Humanin's anti-cell death activity in some cultured neuronal cells. However, it remains unknown whether SH3BP5 is the sole effector of the Humanin signaling pathway via htHNR/JAKs/STAT3. Here we show that the Humanin signaling pathway via htHNR/JAKs/STAT3 increased the expression levels of mRNA and protein of Apollon/Bruce, an unusual member of the inhibitors of apoptosis proteins, and that overexpression of Apollon/Bruce inhibits neuronal death, caused by a London-type familial AD-linked mutant (V642I) of amyloid β precursor protein. Overall, the results indicate that expression of Apollon/Bruce is upregulated by Humanin, and Apollon/Bruce could be an effector of Humanin in a context-dependent manner.

Topics: Alzheimer Disease; Animals; Apoptosis; Cells, Cultured; Humans; Inhibitor of Apoptosis Proteins; Intracellular Signaling Peptides and Proteins; Janus Kinases; Mice; Signal Transduction; STAT3 Transcription Factor

Amyloid β-peptide (Aβ) has been implicated as a key molecule in the neurodegenerative cascades of Alzheimer's disease (AD). Humanin (HN) is a secretory peptide that inhibits the neurotoxicity of Aβ. However, the mechanism(s) by which HN exerts its neuroprotection against Aβ-induced AD-like pathological changes and memory deficits are yet to be completely defined. In the present study, we provided evidence that treatment of rats with HN increases the number of dendritic branches and the density of dendritic spines, and upregulates pre- and post-synaptic protein levels; these effects lead to enhanced long-term potentiation and amelioration of the memory deficits induced by Aβ(1-42). HN also attenuated Aβ(1-42)-induced tau hyperphosphorylation, apparently by inhibiting the phosphorylation of Tyr307 on the inhibitory protein phosphatase-2A (PP2A) catalytic subunit and thereby activating PP2A. HN also inhibited apoptosis and reduced the oxidative stress induced by Aβ(1-42). These findings provide novel mechanisms of action for the ability of HN to protect against Aβ(1-42)-induced AD-like pathological changes and memory deficits.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Cognition; Cognition Disorders; Dendrites; Dendritic Spines; Disease Models, Animal; Hippocampus; Intracellular Signaling Peptides and Proteins; Male; Maze Learning; Neurons; Oxidative Stress; Phosphorylation; Rats; Rats, Wistar

Humanin is a secreted bioactive peptide that is protective in a variety of death models, including cell-based neuronal death models related to Alzheimer's disease (AD). To mediate the protective effect in AD-related death models, Humanin signals via a cell-surface receptor that is generally composed of three subunits: ciliary neurotrophic factor receptor α, WSX-1 and gp130 (heterotrimeric Humanin receptor; htHNR). However, the protective effect of Humanin via the htHNR is weak (EC50=1-10 μM); therefore, it is possible that another physiological agonist for this receptor exists in vivo. In the current study, calmodulin-like skin protein (CLSP), a calmodulin relative with an undefined function, was shown to be secreted and inhibit neuronal death via the htHNR with an EC50 of 10-100 pM. CLSP was highly expressed in the skin, and the concentration in circulating normal human blood was ~5 nM. When administered intraperitoneally in mice, recombinant CLSP was transported across the blood-cerebrospinal fluid (CSF)-barrier and its concentration in the CSF reaches 1/100 of its serum concentration at 1 h after injection. These findings suggest that CLSP is a physiological htHNR agonist.

Topics: Alzheimer Disease; Amino Acid Sequence; Animals; Biological Transport; Cell Death; Humans; Injections, Intraperitoneal; Intracellular Signaling Peptides and Proteins; Male; Mice; Middle Aged; Molecular Sequence Data; Neurons; Protein Multimerization; Protein Subunits; Proteins; Receptors, Cell Surface; Recombinant Proteins; Skin

Humanin (HN), a 24-amino acid peptide encoded by the mitochondrial 16S rRNA gene, was discovered by screening a cDNA library from the occipital cortex of a patient with Alzheimer's disease (AD) for a protection factor against AD-relevant insults. Earlier, using the yeast two-hybrid system, we have identified the M-phase phosphoprotein 8 (MPP8) as a binding partner for HN. In the present work, we further confirmed interaction of HN with MPP8 in co-immunoprecipitation experiments and localized an MPP8-binding site in the region between 5 and 12 aa. of HN. We have also shown that an MPP8 fragment (residues 431-560) is sufficient to bind HN. Further studies on functional consequences of the interaction between the potential oncopetide and the oncoprotein may elucidate some aspects of the molecular mechanisms of carcinogenesis.

Topics: Alzheimer Disease; Amino Acids; Binding Sites; Cell Line, Tumor; Cell Transformation, Neoplastic; Gene Library; Humans; Intracellular Signaling Peptides and Proteins; Neurons; Oncogene Proteins; Phosphoproteins; Protein Binding; Protein Interaction Maps

Humanin is a secreted bioactive peptide that suppresses cell toxicity caused by a variety of insults. The neuroprotective effect of Humanin against Alzheimer disease (AD)-related death is mediated by the binding of Humanin to its heterotrimeric Humanin receptor composed of ciliary neurotrophic receptor α, WSX-1, and gp130, as well as the activation of intracellular signaling pathways including a JAK2 and STAT3 signaling axis. Despite the elucidation of the signaling pathways by which Humanin mediates its neuroprotection, the transcriptional targets of Humanin that behaves as effectors of Humanin remains undefined. In the present study, Humanin increased the mRNA and protein expression of SH3 domain-binding protein 5 (SH3BP5), which has been known to be a JNK interactor, in neuronal cells. Similar to Humanin treatment, overexpression of SH3BP5 inhibited AD-related neuronal death, while siRNA-mediated knockdown of endogenous SH3BP5 expression attenuated the neuroprotective effect of Humanin. These results indicate that SH3BP5 is a downstream effector of Humanin. Furthermore, biochemical analysis has revealed that SH3BP5 binds to JNK and directly inhibits JNK through its two putative mitogen-activated protein kinase interaction motifs (KIMs).

Topics: Adaptor Proteins, Signal Transducing; Alzheimer Disease; Amino Acid Motifs; Animals; Cell Death; Cell Line, Tumor; Cells, Cultured; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Janus Kinase 2; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Membrane Proteins; Mice; Mitochondrial Proteins; Neurons; Neuroprotective Agents; Protein Structure, Tertiary

Humanin (HN), a peptide of 24 amino acid residues, suppresses the neuronal cell death that is induced by the gene products of Alzheimer's disease. HN contains two Ser residues at positions 7 and 14. Because the proportion of D-Ser isomerized from L-Ser in proteins appears to increase as cellular organs age, we explored the structural effects of the isomerization of each Ser residue in HN. By using a thioflavin-T assay to detect fibril formation, we found that an HN derivative that contained two isomerized D-Ser residues had a greater tendency to form fibrils than did wild-type HN or HNs containing single D-Ser residues. A previous report showed that HN containing two D-Ser residues exerts neuroprotective activity. Our data, therefore, suggest that the fibril formation by HN that contains two D-Ser residues may promote HN neuroprotective activity.

Topics: Alzheimer Disease; Amino Acid Sequence; Benzothiazoles; Circular Dichroism; Congo Red; Humans; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; Neuroprotective Agents; Protein Structure, Secondary; Solutions; Stereoisomerism; Structure-Activity Relationship; Thiazoles

Because of the immunogenicity and toxicity in vivo of large molecules such as lectins, the application of these molecules is remarkably restricted in drug delivery systems. In this study, to improve the brain drug delivery and reduce the immunogenicity of traditional lectin modified delivery system, Odorranalectin (OL, 1700 Da), a novel non-immunogenic small peptide, was selected to establish an OL-modified cubosomes (Cubs) system. The streptavidin (SA)-conjugated Cubs were prepared by incorporating maleimide-PEG-oleate and taking advantage of its thiol group binding reactivity to conjugate with 2-iminothiolane thiolated SA; mono-biotinylated OL was then coupled with the SA-modified Cubs. The OL-decorated Cubs (OL-Cubs) devised via a non-covalent SA-biotin "bridge" made it easy to conjugate OL and determine the number of ligands on the surface of the Cubs using sensitive chemiluminescent detection. Retention of the bio-recognitive activity of OL after covalent coupling was verified by hemagglutination testing. Nose-to-brain delivery characteristic of OL-Cubs was investigated by in vivo fluorescent biodistribution using coumarin-6 as a marker. The relative uptake of coumarin carried by OL-Cubs was 1.66- to 3.46-fold in brain tissues compared to that incorporated in the Cubs. Besides, Gly14-Humanin (S14G-HN) as a model peptide drug was loaded into cubosomes and evaluated for its pharmacodynamics on Alzheimer's disease (AD) rats following intranasal administration by Morris water maze test and acetylcholinesterase activity determination. The results suggested that OL functionalization enhanced the therapeutic effects of S14G-HN-loaded cubosomes on AD. Thus, OL-Cubs might offer a novel effective and noninvasive system for brain drug delivery, especially for peptides and proteins.

Topics: Administration, Intranasal; Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Coumarins; Disease Models, Animal; Drug Carriers; Drug Delivery Systems; Glycine; Intracellular Signaling Peptides and Proteins; Lectins; Maze Learning; Peptide Fragments; Rats; Rats, Sprague-Dawley; Streptavidin; Thiazoles; Tissue Distribution

Some ocular diseases characterized by apoptotic death of retinal ganglion cells (RGCs) and Alzheimer's disease (AD) are chronic neurodegenerative disorders and have similarities in neuropathology. Humanin (HN) is known for its ability to suppress neuronal death induced by AD-related insults. In present study, we investigated the neuroprotective effects of HN on hypoxia-induced toxicity in RGC-5 cells. Hypoxia mimetic compound cobalt chloride (CoCl₂) could increase the cell viability loss and apoptosis, whereas HN can significantly attenuate these effects. This finding may provide new therapeutics for the retinal neurodegenerative diseases targeting neuroprotection.

Topics: Alzheimer Disease; Animals; Antimutagenic Agents; Apoptosis; Cell Line, Transformed; Cobalt; Intracellular Signaling Peptides and Proteins; Neuroprotective Agents; Rats; Retinal Ganglion Cells

Humanin (HN) is a linear 24-aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß-amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1-40) and characterization of the interaction structure through a molecular modeling study. Wild-type HN and HN-sequence mutations were synthesized by SPPS and the HPLC-purified peptides characterized by MALDI-MS. The interaction epitopes between HN and Aß(1-40) were identified by affinity-MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity-bound peptides. The affinity-MS analyses revealed HN(5-15) as the epitope sequence of HN, whereas Aß(17-28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1-40) and by ELISA with Aß(1-40) and Aß-partial sequences as ligands to immobilized HN. The specificity and affinity of the HN-Aß interaction were characterized by direct ESI-MS of the HN-Aß(1-40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K(D) of the complex of 610 nm. A molecular dynamics simulation of the HN-Aß(1-40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1-40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Intracellular Signaling Peptides and Proteins; Models, Molecular; Neuroprotective Agents; Protein Conformation; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Topics: Alzheimer Disease; Animals; Cell Death; Humans; Intracellular Signaling Peptides and Proteins; Neurons; Receptors, Interleukin-6

Humanin (HN), a 24-residue peptide, was identified as a novel neuroprotective factor and shows anti-cell death activity against a wide spectrum of Alzheimer's disease (AD)-related cytotoxicities, including exposure to amyloid beta (Abeta), in vitro. We previously demonstrated that the injection of S14G-HN, a highly potent HN derivative, into brain ameliorated memory loss in an Abeta-injection mouse model. To fully understand HN's functions under AD-associated pathological conditions, we examined the effect of S14G-HN on triple transgenic mice harboring APP(swe), tau(P310L), and PS-1(M146V) that show the age-dependent development of multiple pathologies relating to AD. After 3 months of intranasal treatment, behavioral analyses showed that S14G-HN ameliorated cognitive impairment in male mice. Moreover, ELISA and immunohistochemical analyses showed that Abeta levels in brains were markedly lower in S14G-HN-treated male and female mice than in vehicle control mice. We also found the expression level of neprilysin, an Abeta degrading enzyme, in the outer molecular layer of hippocampal formation was increased in S14G-HN-treated mouse brains. NEP activity was also elevated by S14G-HN treatment in vitro. These findings suggest that decreased Abeta level in these mice is at least partly attributed to S14G-HN-induced increase of neprilysin level. Although HN was identified as an anti-neuronal death factor, these results indicate that HN may also have a therapeutic effect on amyloid accumulation in AD.

Topics: Age Factors; Alzheimer Disease; Amyloid beta-Peptides; Animals; Brain; Female; Hippocampus; Intracellular Signaling Peptides and Proteins; Male; Memory Disorders; Mice; Mice, Transgenic; Neprilysin

A single mutation has resulted in large differences in neuroprotective activity of a 24 amino acid Humanin (HN). A mutation of Ser7Ala (S7A-HN) resulted in loss of activity, while a mutation of Ser14Gly (S14G-HN) resulted in about 1000-fold increase. The mechanism of the effects conferred by these mutations have been totally unclear, although our recent structure analysis suggested a possibility of the effect of mutation on the structure stability. Here, we have studied the effects of buffer and temperature on the structure of these three HN peptides. These peptides showed a similar disordered structure at 10°C in 10mM phosphate, pH 6.0. They were also similar in phosphate-buffered saline (PBS) as long as the temperature was kept low at 10°C. However, a large difference was observed in both phosphate buffer and PBS between the peptides, when the temperature was raised to a physiological temperature of 37°C. While S14G-HN showed small changes in both solutions at 37°C, the less active HN and inactive S7A-HN showed much larger changes under the identical conditions. In addition, it appeared that structure change at 37°C was faster for S7A-HN than HN. These results show that the structure stability at 37°C increases in the order of S7A-HN, HN and S14G-HN, in correlation with their neuroprotective activities.

Topics: Alzheimer Disease; Circular Dichroism; Drug Discovery; Humans; Hydrogen-Ion Concentration; Intracellular Signaling Peptides and Proteins; Mutation, Missense; Protein Conformation; Structure-Activity Relationship; Temperature

Mitochondrial dysfunction is a hallmark of beta-amyloid (Abeta)-induced neuronal toxicity in Alzheimer's disease (AD), and is considered as an early event in AD pathology. Humanin (HN) and its derivative, [Gly14]-Humanin (HNG), are known for their ability to suppress neuronal death induced by AD-related insults in vitro and in vivo. In the present study, we investigated the neuroprotective effects of HNG on Abeta(25-35)-induced toxicity and its potential mechanisms in PC12 cells. Exposure of PC12 cells to 25 microM Abeta(25-35) caused significant viability loss and cell apoptosis. In addition, decreased mitochondrial membrane potential and increased cytochrome c releases from mitochondria were also observed after Abeta(25-35) exposure. All these effects induced by Abeta(25-35) were markedly reversed by HNG. Pretreatment with 100 nM HNG 6h prior to Abeta(25-35) exposure significantly elevated cell viability, reduced Abeta(25-35)-induced cell apoptosis, stabilized mitochondrial membrane potential, and blocked cytochrome c release from mitochondria. Furthermore, HNG also ameliorated the Abeta(25-35)-induced Bcl-2/Bax ratio reduction and decreased caspase-3 activity in PC12 cells. These results demonstrate that HNG could attenuate Abeta(25-35)-induced PC12 cell injury and apoptosis by preventing mitochondrial dysfunction. Furthermore, these data suggest that mitochondria are involved in the protective effect of HNG against Abeta(25-35).

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Survival; Cytoprotection; Energy Metabolism; Intracellular Signaling Peptides and Proteins; Membrane Potentials; Metabolic Networks and Pathways; Mitochondria; Mitochondrial Diseases; Nerve Degeneration; Neuroprotective Agents; PC12 Cells; Peptide Fragments; Rats

Decline in insulin action is a metabolic feature of aging and is involved in the development of age-related diseases including Type 2 Diabetes Mellitus (T2DM) and Alzheimer's disease (AD). A novel mitochondria-associated peptide, Humanin (HN), has a neuroprotective role against AD-related neurotoxicity. Considering the association between insulin resistance and AD, we investigated if HN influences insulin sensitivity.. Using state of the art clamp technology, we examined the role of central and peripheral HN on insulin action. Continuous infusion of HN intra-cerebro-ventricularly significantly improved overall insulin sensitivity. The central effects of HN on insulin action were associated with activation of hypothalamic STAT-3 signaling; effects that were negated by co-inhibition of hypothalamic STAT-3. Peripheral intravenous infusions of novel and potent HN derivatives reproduced the insulin-sensitizing effects of central HN. Inhibition of hypothalamic STAT-3 completely negated the effects of IV HN analog on liver, suggesting that the hepatic actions of HN are centrally mediated. This is consistent with the lack of a direct effect of HN on primary hepatocytes. Furthermore, single treatment with a highly-potent HN analog significantly lowered blood glucose in Zucker diabetic fatty rats. Based upon the link of HN with two age-related diseases, we examined if there were age associated changes in HN levels. Indeed, the amount of detectable HN in hypothalamus, skeletal muscle, and cortex was decreased with age in rodents, and circulating levels of HN were decreased with age in humans and mice.. We conclude that the decline in HN with age could play a role in the pathogenesis of age-related diseases including AD and T2DM. HN represents a novel link between T2DM and neurodegeneration and along with its analogues offers a potential therapeutic tool to improve insulin action and treat T2DM.

Topics: Alzheimer Disease; Animals; Blood Glucose; Diabetes Mellitus, Type 2; Insulin; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Liver; Male; Muscle, Skeletal; Rats; Rats, Sprague-Dawley; Rats, Zucker

Humanin (HN) inhibits Alzheimer's disease (AD)-relevant neuronal death and dysfunction, by interacting with a receptor (s) involving ciliary neurotrophic factor receptor alpha (CNTFR), WSX-1, and gp130. It remains unknown whether this complex is the sole HN receptor that mediates HN-induced anti-AD activity. We here report that an alternatively spliced WSX-1 isoform, encoding an extracellular 270-amino-acid region of WSX-1 with cytokine-binding regions (named soluble WSX-1; sWSX-1), is expressed in neuronal cells lacking function of full-length WSX-1 and enables HN to rescue AD-relevant death. This result suggests that CNTFR/soluble WSX-1/gp130 behaves as an alternative functional HN receptor.

Topics: Alternative Splicing; Alzheimer Disease; Amino Acid Sequence; Animals; Apoptosis; Base Sequence; Ciliary Neurotrophic Factor Receptor alpha Subunit; Exons; Intracellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Molecular Sequence Data; Neurons; Phosphorylation; Receptors, Cytokine; Receptors, Interleukin; STAT3 Transcription Factor

The structural and dynamical properties of Humanin, a small peptide with neuroprotective activity against the insults of the Alzheimer's disease-related genes and the neurotoxic amyloid peptide, are studied in two different environments by molecular dynamics simulation. In this study, we have performed comparative molecular dynamics simulations in the absence and in the presence of TFE. The resulting trajectories were analyzed in terms of structural and dynamical properties of peptide and compared to the available NMR data. In water humanin is observed to partly unfold. The peptide is readily stabilized in an ordered helical conformation in the TFE/water mixture. Our simulations show that the peptide is flexible with definite turn point in its structure in water environment. It is free to interact with receptors that mediate its action in polar environment. Humanin may also find an alpha helix structure necessary for passage through biomembranes and/or specific interactions.

Topics: Alzheimer Disease; Computer Simulation; Humans; Hydrogen Bonding; Intracellular Signaling Peptides and Proteins; Models, Molecular; Neuroprotective Agents; Protein Structure, Secondary; Trifluoroethanol; Water

Topics: Administration, Intranasal; Alzheimer Disease; Animals; Diabetes Mellitus; Drug Delivery Systems; Drug Design; Fibroblast Growth Factors; Humans; Intracellular Signaling Peptides and Proteins; Osteoporosis; Parathyroid Hormone; Peptide Fragments; Peptides; Proteins; Psoriasis

The structure of a highly potent Ser14Gly analog of antiAlzheimer peptide, Humanin, was examined by circular dichroism (CD). The secondary structure is more disordered in water than in phosphate-buffered saline (PBS). The peptide structure in water is little dependent on both peptide concentration and temperature. On the contrary, the peptide structure was significantly different in PBS from the structure in water, which is more apparent at a higher peptide concentration and temperature. The observed different structure in PBS appears to be due to self-association of the peptide, which is enhanced by elevated temperature and, hence, via hydrophobic interactions. The wild-type Humanin also behaved similarly, i.e., it assumed a disordered structure in water but underwent conformational changes in PBS. Although high peptide concentrations for CD measurements are not encountered in vivo, the results suggest the tendency of the peptide to interact hydrophobically with other structures as well as with itself.

Topics: Alzheimer Disease; Circular Dichroism; Glycine; Humans; Hydrophobic and Hydrophilic Interactions; Intracellular Signaling Peptides and Proteins; Mutation; Protein Structure, Secondary; Serine; Solubility; Temperature; Ultraviolet Rays

The NMR solution study of Ser14Gly-humanin (S14G-HN), a 1000-fold more potent derivative of humanin (HN), is reported. HN is 24-residue peptide that selectively suppresses neuronal cell death caused by Alzheimer's disease (AD)-specific insults and offers hope for the development of a cure against AD. In aqueous solution the NMR data show that S14G-HN is a flexible peptide with turn-like structures in its conformational ensemble distributed over an extensive part of its sequence from Pro3 to Glu15. In the more lipophilic environment of 30% TFE, an alpha-helical structure spanning residues Phe6 to Thr13 is identified. Comparison of these findings to the NMR structure of the parent HN and to existing structure-function relationship literature data outlines the important for activity structural features for this class of neuroprotective peptides, and brings forth flexibility as an important characteristic that may facilitate interactions with functional counterparts of the neuroprotection pathway.

Topics: Alzheimer Disease; Amino Acid Sequence; Apoptosis; Base Sequence; Circular Dichroism; Glycine; Humans; Intracellular Signaling Peptides and Proteins; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Neurons; Neuroprotective Agents; Protein Structure, Secondary; Serine

Alzheimer's disease (AD) is the leading cause of dementia for aging people, and far from control due to its obscure mechanism. Humanin, a 24-aa peptide encoded by a newly identified gene cloned from an apparently normal region of AD brain, can specifically attenuate AD-related neurotoxicity. It protects neurons from insults of various AD genes, anti-APP antibodies and Abeta by forming a homodimer outside and interfering directly or indirectly with the activity of Abeta. Humanin seems, however, not to inhibit other toxic insults to neurons, such as Fas or etoposide, an agent against carcinomatous cells in clinical therapy. So Humanin rescues neurons from various AD-related toxicity specifically with efficiency.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Humans; Intracellular Signaling Peptides and Proteins; Neuroprotective Agents; Peptides

Humanin is a newly identified 24-residue peptide that suppresses neuronal cell death caused by a wide spectrum of familial Alzheimer's disease genes and the beta-amyloid peptide. In this study, NMR and circular dichroism studies of synthetic humanin in aqueous and 30% 2,2,2-trifluoroethanol (TFE) solutions are reported. In aqueous solution, humanin exists predominantly in an unstructured conformation in equilibrium with turn-like structures involving residues Gly5 to Leu10 and Glu15 to Leu18, providing indication of nascent helix. In the less polar environment of 30% TFE, humanin readily adopts helical structure with long-range order spanning residues Gly5 to Leu18. Comparative 3D modeling studies and topology predictions are in qualitative agreement with the experimental findings in both environments. Our studies reveal a flexible peptide in aqueous environment, which is free to interact with possible receptors that mediate its action, but may also acquire a helical conformation necessary for specific interactions and/or passage through membranes.

Topics: Alzheimer Disease; Circular Dichroism; Glutamic Acid; Glycine; Humans; Intracellular Signaling Peptides and Proteins; Leucine; Magnetic Resonance Spectroscopy; Models, Molecular; Peptides; Protein Conformation; Protein Structure, Secondary; Proteins; Temperature; Trifluoroethanol; Water

The basis of a bacterial pathogenic process consists in the change of a certain host structure to a completely different one. This is accomplished by binding of a bacterial protein product to the host structure. Streptococcal NAD+-nucleosidase was explored as to its binding to the host receptor represented by beef heart extract. The bacterial product was found to bind to the host structure until the available host structure was fully saturated. The similarity of the above flows of macromolecules with some models of morphogenesis indicates the existence of diseases associated with the flow of a protein to the undesirable site in the organism. In such a case therapy with low-molar-mass substances is wrong in principle.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Bacterial Proteins; Biomarkers, Tumor; Humans; Intracellular Signaling Peptides and Proteins; Models, Biological; Myocardium; NAD+ Nucleosidase; Neoplasms; Peptides; Protein Binding; Streptococcus pyogenes

Humanin (HN) inhibits neuronal cell death induced by various Alzheimer's disease (AD)-related insults. It has been proposed that HN binds to a putative receptor on the cell membrane and triggers a signal transduction cascade linked to neuroprotection. Recently, it was shown that HN binds to pertussis toxin (PTX)-sensitive G protein-coupled formylpeptide receptor-like-1 molecule (FPRL-1), reduces A beta(1--42) aggregation and fibril formation, and suppresses the A beta(1--42) toxicity on mononuclear phagocytic cells [Ying, G., Iribarren, P., Zhou, Y., Gong, W., Zhang, N., Yu, Z.X., Le, Y., Cui, Y., Wang, J.M., 2004. Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor. Journal of Immunology 172 (11), 7078--7085.]. We here show that siRNA-mediated disruption of expression of the mouse counterpart of FPRL-1, FPR2, did not result in attenuation of HN-mediated rescue of neuronal cell death induced by AD-related insults. We simultaneously provide evidence that neuroprotection by HN in F11 cells is mediated by the STAT3 transcription factor as well as by certain tyrosine kinases. Altogether, we speculate that a receptor other than FPR2 exists that mediates HN neuroprotection in F11 neurohybrid cells.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antineoplastic Agents; Apoptosis; DNA-Binding Proteins; Genistein; Humans; Intracellular Signaling Peptides and Proteins; Mice; Neurons; Neuroprotective Agents; Protein-Tyrosine Kinases; Receptors, Formyl Peptide; RNA, Small Interfering; STAT3 Transcription Factor; Trans-Activators

Alzheimer's disease (AD) is the most common cause of dementia. Humanin (HN) is a short bioactive peptide abolishing neuronal cell death induced by various familial AD (FAD)-causative genes and amyloid-beta (Abeta) in vitro. It has been shown that HN suppresses memory impairment of mice induced by intracerebroventricular administration of Abeta. To potentiate the neuroprotective effect of HN, we synthesized a hybrid peptide named Colivelin composed of activity-dependent neurotrophic factor (ADNF) C-terminally fused to AGA-(C8R)HNG17, a potent HN derivative. Colivelin completely suppresses death induced by overexpressed FAD-causative genes and Abeta1-43 at a concentration of 100 fM, whereas AGA-(C8R)HNG17 does so at a concentration of 10 pM. Colivelin-induced neuroprotection has been confirmed to occur via two neuroprotective pathways: one mediated by Ca2+/calmodulin-dependent protein kinase IV, triggered by ADNF, and one mediated by signal transducer and activator of transcription 3, triggered by HN. In vivo animal studies have further indicated that intracerebroventricular administration of Colivelin not only completely suppresses impairment in spatial working memory induced by repetitive intracerebroventricular injection of Abeta25-35 or Abeta1-42, but also it antagonizes neuronal loss in the CA1 region of hippocampus induced by hippocampal injection of Abeta1-42. In addition, intraperitoneally administered Colivelin suppresses memory impairment caused by a muscarinic acetylcholine receptor antagonist, 3-quinuclidinyl benzilate, indicating that a substantial portion of intraperitoneally administered Colivelin passes through the blood-brain barrier and suppresses functional memory deficit. Thus, Colivelin might serve as a novel drug candidate for treatment of AD.

Topics: Alzheimer Disease; Amino Acid Sequence; Animals; Cell Death; Cell Line; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nerve Tissue Proteins; Neuroprotective Agents; Peptide Fragments; Peptides; Rats

Alzheimer's disease (AD) is characterized by overproduction of beta amyloid peptides in the brain with progressive loss of neuronal cells. The 42-aa form of the beta amyloid peptide (Abeta(42)) is implied as a major causative factor, because it is toxic to neurons and elicits inflammatory responses in the brain by activating microglial cells. Despite the overproduction of Abeta(42), AD brain tissue also generates protective factor(s) that may antagonize the neurodestructive effect of Abeta(42). Humanin is a gene cloned from an apparently normal region of an AD brain and encodes a 24-aa peptide. Both secreted and synthetic Humanin peptides protect neuronal cells from damage by Abeta(42), and the effect of Humanin may involve putative cellular receptor(s). To elucidate the molecular identity of such receptor(s), we examined the activity of synthetic Humanin on various cells and found that Humanin induced chemotaxis of mononuclear phagocytes by using a human G protein-coupled formylpeptide receptor-like-1 (FPRL1) and its murine counterpart FPR2. Coincidentally, FPRL1 and FPR2 are also functional receptors used by Abeta(42) to chemoattract and activate phagocytic cells. Humanin reduced the aggregation and fibrillary formation by suppressing the effect of Abeta(42) on mononuclear phagocytes. In neuroblast cells, Humanin and Abeta(42) both activated FPRL1; however, only Abeta(42) caused apoptotic death of the cells, and its cytopathic effect was blocked by Humanin. We conclude that Humanin shares human FPRL1 and mouse FPR2 with Abeta(42) and suggest that Humanin may exert its neuroprotective effects by competitively inhibiting the access of FPRL1 to Abeta(42).

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Humans; Intracellular Signaling Peptides and Proteins; Mice; Neurons; Neuroprotective Agents; Proteins; Receptors, Formyl Peptide; Receptors, Lipoxin

Presenilin (PS)1 and its mutants, which consist of the N-terminal and C-terminal fragments, cause certain familial forms of Alzheimer's disease (FAD). Our earlier studies found that FAD-linked M146L-PS1 causes neuronal cell death through nitrogen oxide synthase (NOS) and that FAD-linked N141I-PS2, another member of the PS family, causes neuronal cell death through NADPH oxidase. In this study, we examined 27 different FAD-linked mutants of PS1, and found that PS1 mutants with mutations in the N-terminal fragment caused NOS inhibitor (NOSI)-sensitive neuronal cell death; in contrast, the PS1 mutants with mutations in the C-terminal fragment caused NOSI-resistant neuronal cell death. The former toxicity was resistant to the specific NADPH oxidase inhibitor apocynin and was inhibited by Humanin (HN), a newly identified neuroprotective factor against Alzheimer's disease (AD)-relevant insults, but not by insulin-like growth factor-I (IGF-I). In contrast, the latter toxicity was sensitive to apocynin and inhibited by both IGF-I and HN. This study indicates for the first time that N- and C-terminal fragment PS1 mutants can generate distinct neurotoxic signals, which will provide an important clue to the understanding of the entire array of neurotoxic signals generated by FAD-causative mutations of PS1.

Topics: Acetophenones; Alzheimer Disease; Animals; Cell Death; Cells, Cultured; Enzyme Inhibitors; Immunoblotting; Insulin-Like Growth Factor I; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mutation; NADPH Oxidases; Neurons; Neuroprotective Agents; Neurotoxins; Nitric Oxide Synthase; Presenilin-1; Proteins

Amyloid precursor protein (AbetaPP), a precursor of amyloid beta (Abeta) peptide, is one of the molecules involved in the pathogenesis of Alzheimer's disease (AD). Specific mutations in AbetaPP have been found in patients inheriting familial AD (FAD). These mutant AbetaPP proteins cause cell death in neuronal cell lines in vitro, but the molecular mechanism of cytotoxicity has not yet been clarified completely. We analyzed the cytotoxic mechanisms of the London-type AbetaPP mutant, V642I-AbetaPP, in primary cortical neurons utilizing an adenovirus-mediated gene transfer system. Expression of V642I-AbetaPP protein induced degeneration of the primary neurons. This cytotoxicity was blocked by pertussis toxin, a specific inhibitor for heterotrimeric G proteins, Go/i, and was suppressed by an inhibitor of caspase-3/7 and an antioxidant, glutathione ethyl ester. A specific inhibitor for NADPH oxidase, apocynin, but not a xanthine oxidase inhibitor or a nitric oxide inhibitor, blocked V642I-AbetaPP-induced cytotoxicity. Among mitogen-activated protein kinase (MAPK) family proteins, c-Jun N-terminal kinase (JNK) and p38MAPK, but not extracellular regulated kinase (ERK), were involved in this cytotoxic pathway. The V642I-AbetaPP-induced cytotoxicity was not suppressed by two secretase inhibitors, suggesting that Abeta does not play a major role in this cytotoxicity. Two neuroprotective factors, insulin-like growth factor I (IGF-I) and Humanin, protected these primary neurons from V642I-AbetaPP-induced cytotoxicity. Furthermore, interleukin-6 and -11 also attenuated this cytotoxicity. This study demonstrated that the signaling pathway activated by mutated AbetaPP in the primary neurons is the same as that by the other artificial insults such as antibody binding to AbetaPP and the artificial dimerization of cytoplasmic domain of AbetaPP. The potential of neurotrophic factors and cytokines in AD therapy is also indicated.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Caspase Inhibitors; Caspases; Cells, Cultured; Enzyme Inhibitors; Fetus; Genetic Vectors; GTP-Binding Protein alpha Subunits, Gi-Go; Insulin-Like Growth Factor I; Interleukin-11; Interleukin-6; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; Mutation; NADPH Oxidases; Nerve Degeneration; Neurons; Neuroprotective Agents; Neurotoxins; p38 Mitogen-Activated Protein Kinases; Protein Binding; Proteins; Transfection

One of the most important pathological features of Alzheimer's disease (AD) is extracellular senile plaques, whose major component is amyloid-beta peptides (Abeta). Abeta binds to the extracellular domain of p75NTR (p75 neurotrophin receptor) and induces neuronal cell death. We investigated the molecular mechanism of Abeta-induced neurotoxicity in detail from the standpoint of interaction between p75NTR and its recently identified relative, PLAIDD (p75-like apoptosis-inducing death domain). Using F11 neuronal hybrid cells, we demonstrate that there are two distinct pathways for Abeta-induced toxicity mediated by p75NTR. One pathway that has been previously elucidated, is mediated by p75NTR, Go, JNK, NADPH oxidase and caspase3-related caspases. We found that PLAIDD and Gi proteins, heterotrimeric G proteins, are involved in the alternative Abeta-induced neurotoxicity mediated by p75NTR. The alternative pathway triggered by Abeta is thus mediated by p75NTR, PLAIDD, Gi, JNK, NADPH oxidase and caspase3-related caspases. In addition, we found that HN, ADNF, IGF-I, or bFGF inhibits both pathways of Abeta-induced neurotoxicity mediated by p75NTR.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Apoptosis Regulatory Proteins; Carrier Proteins; Caspase 3; Caspases; Cell Death; Cell Line; GTP-Binding Protein alpha Subunits, Gi-Go; Hybrid Cells; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Membrane Proteins; Mice; Mitogen-Activated Protein Kinases; Mutagenesis, Site-Directed; NADPH Oxidases; Neurons; Peptide Fragments; Pertussis Toxin; Proteins; Rats; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Signal Transduction; Transfection

Although neurotoxic functions are well characterized in familial Alzheimer's disease (FAD)-linked N141I mutant of presenilin (PS)2, little has been known about M239V-PS2, another established FAD-causative mutant. We found that expression of M239V-PS2 caused neuronal cytotoxicity. M239V-PS2 exerted three forms of cytotoxicity: one was sensitive to both an antioxidant glutathione-ethyl-ester (GEE) and a caspase inhibitor Ac-DEVD-CHO (DEVD); the second was sensitive to GEE but resistant to DEVD; and the third was resistant to both. The GEE/DEVD-sensitive cytotoxicity by M239V-PS2 was likely through NADPH oxidase and the GEE-sensitive/DEVD-resistant cytotoxicity through xanthine oxidase (XO). Both mechanisms by M239V-PS2 were suppressed by pertussis toxin (PTX) and were mediated by Galpha(o), but not by Galpha(i). Although Abeta1-43 itself induced no cytotoxicity, Abeta1-43 potentiated all three components of M239V-PS2 cytotoxicity. As these cytotoxic mechanisms by M239V-PS2 are fully shared with N141I-PS2, they are most likely implicated in the pathomechanism of FAD by PS2 mutations. Notably, cytotoxicity by M239V-PS2 could be inhibited by the combination of two clinically usable inhibitors of superoxide-generating enzymes, apocynin and oxypurinol.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antioxidants; Enzyme Inhibitors; GTP-Binding Protein alpha Subunits, Gi-Go; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mutation; NADPH Oxidases; Nerve Degeneration; Neurotoxins; Peptide Fragments; Presenilin-2; Proteins; Rats; Tumor Cells, Cultured; Xanthine Oxidase

Humanin is a novel, 24-mer residue bioactive peptide, which antagonizes Alzheimer's disease (AD) related neurotoxicity and offers a hope for developing new therapeutics against AD. Access to adequate amounts of pure humanin is a prerequisite for further, thorough, investigation of the pharmacological properties and therapeutic potency of the peptide. Until now, humanin has been obtained mainly by molecular biology techniques. In this work the Fmoc solid-phase synthesis of humanin on an in-house prepared 2-Cl-tritylamidomethyl polystyrene resin is described fully. Special precautions, i.e. prolonged deprotection steps, should be taken to achieve a high overall yield, since humanin seems to contain a 'difficult sequence' (R4G5F6S7C8L9) near its highly lipophilic, biologically important region L9L10L11L12.

Topics: Alzheimer Disease; Combinatorial Chemistry Techniques; Humans; Intracellular Signaling Peptides and Proteins; Neuroprotective Agents; Peptides; Proteins

Neuronal cell death accounts for the clinical manifestations in Alzheimer's disease (AD). To establish the curative therapy of AD, neuroprotection is one of the primary therapeutic targets, and the elucidation of the mechanism of neuronal cell death is mandatory. Detailed characterization of neuronal cell death caused by familial AD (FAD)-linked mutant genes revealed that different cell death pathways are evoked by different types of mutants. Humanin (HN), a newly identified neuroprotective peptide, suppresses neuronal cell death caused by all known FAD mutants and A beta, while it has no effect on neuronal cell death caused by AD-irrelevant insults. The functional target of HN is the antagonism to neuronal death, not the modulation of A beta production, suggesting that HN-based medication can be combined with other remedies targeting A beta. HN is a promising seed for a novel therapy aiming at complete cure of AD through the suppression of neuronal loss.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Apoptosis; Brain; Cell Death; Cell Survival; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Presenilin-1; Presenilin-2; Proteins

Humanin (HN) is a newly identified neuroprotective peptide that specifically suppresses Alzheimer's disease (AD)-related neurotoxicity. HN peptide has been detected in the human AD brain as well as in mouse testis and colon by immunoblot and immunohistochemical analyses. By means of yeast two-hybrid screening, we identified TRIM11 as a novel HN-interacting protein. TRIM11, which is a member of protein family containing a tripartite motif (TRIM), is composed of a RING finger domain, which is a putative E3 ubiquitin ligase, a B-box domain, a coiled-coil domain and a B30.2 domain. Deletion of the B30.2 domain in TRIM11 abolished the interaction with HN, whereas the B30.2 domain alone did not interact with HN. For their interaction, at least the coiled-coil domain was indispensable together with the B30.2 domain. The intracellular level of glutathione S-transferase-fused or EGFP-fused HN peptides or plain HN was drastically reduced by the coexpression of TRIM11. Disruption of the RING finger domain by deleting the first consensus cysteine or proteasome inhibitor treatment significantly diminished the effect of TRIM11 on the intracellular level of HN. These results suggest that TRIM11 plays a role in the regulation of intracellular HN level through ubiquitin-mediated protein degradation pathways.

Topics: Adaptor Proteins, Signal Transducing; Alzheimer Disease; Animals; Carrier Proteins; Immunoblotting; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Neuroprotective Agents; Polymerase Chain Reaction; Proteins; Two-Hybrid System Techniques; Ubiquitin; Yeasts

The 24-residue peptide Humanin (HN), containing two Ser residues at positions 7 and 14, protects neuronal cells from insults of various Alzheimer's disease (AD) genes and A beta. It was not known why the rescue function of (S14G)HN is more potent than HN by two to three orders of magnitude. Investigating the possibility that the post-translational modification of Ser14 might play a role, we found that HN with D-Ser at position 14 exerts neuroprotection more potently than HN by two to three orders of magnitude, whereas D-Ser7 substitution does not affect the rescue function of HN. On the other hand, S7A substitution nullified the HN function. Multiple series of experiments indicated that Ser7 is necessary for self-dimerization of HN, which is essential for neuroprotection by this factor. These findings indicate that the rescue function of HN is quantitatively modulated by d-isomerization of Ser14 and Ser7-relevant dimerization, allowing for the construction of a very potent HN derivative that was fully neuroprotective at 10 pM against 25 microM A beta1-43. This study provides important clues to the understanding of the neuroprotective mechanism of HN, as well as to the development of novel AD therapeutics.

Topics: Alzheimer Disease; Amino Acid Substitution; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Cell Death; Cells, Cultured; Dimerization; Dose-Response Relationship, Drug; Drug Synergism; Hybrid Cells; Intracellular Signaling Peptides and Proteins; Isomerism; Mice; Neurons; Neuroprotective Agents; Phosphorylation; Proteins; Rats; Recombinant Fusion Proteins; Serine; Structure-Activity Relationship; Transfection

The biological function of full-length amyloid-beta protein precursor (AbetaPP), the precursor of Abeta, is not fully understood. Multiple laboratories have reported that antibody binding to cell surface AbetaPP causes neuronal cell death. Here we examined whether induced dimerization of the cytoplasmic domain of AbetaPP (AbetaPPCD) triggers neuronal cell death. In neurohybrid cells expressing fusion constructs of the epidermal growth factor (EGF) receptor with AbetaPPCD (EGFR/AbetaPP hybrids), EGF drastically enhanced neuronal cell death in a manner sensitive to acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-aldehyde (Ac-DEVD-CHO; DEVD), GSH-ethyl ester (GEE), and pertussis toxin (PTX). Dominant-negative apoptosis signal-regulating kinase 1 (ASK1) blocked this neuronal cell death, but not alpha-synuclein-induced cell death. Constitutively active ASK1 (caASK1) caused DEVD/GEE-sensitive cell death in a manner resistant to PTX and sensitive to Humanin, which also suppressed neuronal cell death by EGFR/AbetaPP hybrid. ASK1 formed a complex with AbetaPPCD via JIP-1b, the c-Jun N-terminal kinase (JNK)-interacting protein. EGFR/AbetaPP hybrid-induced and caASK1-induced neuronal cell deaths were specifically blocked by SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one), a specific JNK inhibitor. Combined with our earlier study, these data indicate that dimerization of AbetaPPCD triggers ASK1/JNK-mediated neuronal cell death. We also noticed a potential role of ASK1/JNK in sustaining the activity of this mechanism after initial activation by AbetaPP, which allows for the achievement of cell death by short-term anti-AbetaPP antibody treatment. Understanding the function of AbetaPPCD and its downstream pathway should lead to effective anti-Alzheimer's disease therapeutics.

Topics: Adaptor Proteins, Signal Transducing; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Anthracenes; Carrier Proteins; Dimerization; Epidermal Growth Factor; ErbB Receptors; Flavonoids; Humans; Hybrid Cells; Imidazoles; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase Kinase 5; MAP Kinase Kinase Kinases; Mice; Mitogen-Activated Protein Kinases; Neurons; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Protein Structure, Tertiary; Proteins; Pyridines; Rats; Signal Transduction

Humanin (HN) is a secretory peptide that inhibits neurotoxicity by various Alzheimer's disease-relevant insults. We have so far identified that the substitution of Leu9 for Arg nullifies the extracellular secretion of HN. Here we comprehensively investigate the amino acid requirement of HN essential for its secretion and for its neuroprotective function. Intracellulary expressed HN-EGFP (EGFP N-terminally fused with HN) was extracellularly secreted, whereas neither EGFP nor (L9R)HN-EGFP was secreted at all. While Ala substitution of neither residue affected HN secretion, Arg substitution revealed that the two structures-Leu9-Leu11 and Pro19-Va120-were essential for the secretion of full-length HN. In the Leu9-Leu11 domain, the Leu10 residue turned out to play a central role in this function, because the Asp substitution of Leu10, but not Leu9 or Leu11, nullified the secretion of HN. Utilizing Ala-scanned HN constructs, we also investigated a comprehensive structure-function relationship for the neuroprotective function of full-length HN, which revealed (i) that Pro3, Ser7, Cys8, Leu9, Leu12, Thr13, Ser14, and Pro19 were essential for this function and (ii) that Ser7 and Leu9 were essential for self-dimerization of HN. These findings indicate that HN has activity similar to a signal peptide, for which the Leu9-Leu11 region, particularly Leu10, functions as a core domain, and suggest that self-dimerization of HN is a process essential for its neuroprotective function.

Topics: Alanine; Alzheimer Disease; Amino Acids; Amyloid beta-Protein Precursor; Animals; Arginine; Culture Media, Conditioned; Dimerization; DNA, Complementary; Dose-Response Relationship, Drug; Green Fluorescent Proteins; Hybrid Cells; Intracellular Signaling Peptides and Proteins; Leucine; Luminescent Proteins; Mice; Mutation; Neurons; Neuroprotective Agents; Peptides; Protein Structure, Tertiary; Proteins; Rats; Structure-Activity Relationship; Time Factors; Transfection

Neuronal pathology of the brain with Alzheimer's disease (AD) is characterized by numerous depositions of amyloid-beta peptides (Abeta). Abeta binding to the 75-kDa neurotrophin receptor (p75NTR) causes neuronal cell death. Here we report that Abeta causes cell death in neuronal hybrid cells transfected with p75NTR, but not in nontransfected cells, and that p75NTR(L401K) cannot mediate Abeta neurotoxicity. We analyzed the cytotoxic pathway by transfecting pertussis toxin (PTX)-resistant G protein alpha subunits in the presence of PTX and identified that Galpha(o), but not Galpha(i), proteins are involved in p75NTR-mediated Abeta neurotoxicity. Further investigation suggested that Abeta neurotoxicity via p75NTR involved JNK, NADPH oxidase, and caspases-9/3 and was inhibited by activity-dependent neurotrophic factor, insulin-like growth factor-I, basic fibroblast growth factor, and Humanin, as observed in primary neuron cultures. Understanding the Abeta neurotoxic mechanism would contribute significantly to the development of anti-AD therapies.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Caspases; Cell Death; Fibroblast Growth Factor 2; GTP-Binding Protein alpha Subunits, Gi-Go; Heterotrimeric GTP-Binding Proteins; Humans; Hybrid Cells; Immunoblotting; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; MAP Kinase Kinase 4; Mice; Mitogen-Activated Protein Kinase Kinases; Mutation; NADPH Oxidases; Nerve Tissue Proteins; Neurons; Neuropeptides; Oligopeptides; Proteins; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Somatomedins; Transfection

Insulin-like growth factor-binding protein-3 (IGFBP-3) regulates IGF bioactivity and also independently modulates cell growth and survival. By using a yeast two-hybrid screen to identify IGFBP-3-interacting proteins, we cloned humanin (HN) as an IGFBP-3-binding partner. HN is a 24-aa peptide that has been shown to specifically inhibit neuronal cell death induced by familial Alzheimer's disease mutant genes and amyloid-beta (Abeta). The physical interaction of HN with IGFBP-3 was determined to be of high affinity and specificity and was confirmed by yeast mating, displaceable pull-down experiments with (His)-6-tagged HN, and ligand blot experiments. Co-immunoprecipitation of IGFBP-3 and HN from mouse testes confirmed the interaction in vivo. In cross-linking experiments, HN bound IGFBP-3 but did not compete with IGF-I-IGFBP-3 binding; competitive ligand dot blot experiments revealed the 18-aa heparin-binding domain of IGFBP-3 as the binding site for HN. Alanine scanning determined that F6A-HN mutant does not bind IGFBP-3. HN but not F6A-HN inhibited IGFBP-3-induced apoptosis in human glioblastoma-A172. In contrast, HN did not suppress IGFBP-3 response in SH-SY5Y neuroblastoma and mouse cortical primary neurons. In primary neurons, IGFBP-3 markedly potentiated HN rescue ability from Abeta1-43 toxicity. In summary, we have identified an interaction between the survival peptide HN and IGFBP-3 that is pleiotrophic in nature and is capable of both synergistic and antagonistic interaction. This interaction may prove to be important in neurological disease processes and could provide important targets for drug development.

Topics: Alanine; Alzheimer Disease; Amino Acid Sequence; Apoptosis; Cell Survival; Humans; Insulin-Like Growth Factor Binding Protein 3; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Open Reading Frames; Proteins; Recombinant Proteins; Saccharomyces cerevisiae

Humanin (HN) has been reported to be an endogenous peptide that exerts highly selective neuroprotection against cell death induced by various types of Alzheimer's disease-related insults. We previously proposed the much broader cytoprotective potential of HN from the result that HN suppressed serum-deprivation-induced death of rat pheochromocytoma cells. In this study, we showed that HN also suppressed death of human lymphocytes cultured under serum-deprived condition. Further, we revealed, by assaying metabolic activity and survival rate, that HN was a potent factor capable of increasing the metabolic activity of individual serum-deprived lymphocytes. To our knowledge, there is no report described about a rescue factor that increases the metabolic activity of individual serum-deprived cells and prolongs their survival. This novel feature of HN may enable us to apply this peptide for the management of diseases involving poor metabolic activity, such as mitochondria-related disorders and brain ischemia.

Topics: Adult; Alzheimer Disease; Apoptosis; Cell Death; Cell Line, Tumor; Cell Survival; Cells, Cultured; Culture Media, Serum-Free; DNA; DNA Fragmentation; DNA, Mitochondrial; Dose-Response Relationship, Drug; Genistein; Humans; Intracellular Signaling Peptides and Proteins; Leukocytes, Mononuclear; Lymphocytes; Peptides; Polymerase Chain Reaction; Proteins; Time Factors

An unbiased functional screening with brain cDNA library from an Alzheimer's disease (AD) brain identified a novel 24-residue peptide Humanin (HN), which suppresses AD-related neurotoxicity. As the 1567-base cDNA containing the open reading frame (ORF) of HN is 99% identical to mitochondrial 16S ribosomal RNA as well as registered human mRNA, it was elusive whether HN is produced in vivo. Here, we raised anti-HN antibody and found that long cDNAs containing the ORF of HN (HN-ORF) produced the HN peptide in mammalian cells, dependent on the presence of full-length HN-ORF. Immunoblot analysis detected a 3-kDa protein with HN immunoreactivity in the testis and the colon in 3-week-old mice and in the testis in 12-week-old mice. HN immunoreactivity was also detected in an AD brain, but little in normal brains. This study suggests that HN peptide could be produced in vivo, and would provide a novel insight into the pathophysiology of AD.

Topics: Alzheimer Disease; Animals; Brain; Colon; DNA, Complementary; HeLa Cells; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Lipid Metabolism; Male; Mice; Neurons; Neuroprotective Agents; Open Reading Frames; Proteins; RNA, Ribosomal; Testis

We report a novel gene, designated Humanin (HN) cDNA, that suppresses neuronal cell death by K595N/M596L-APP (NL-APP), a mutant causing familial Alzheimer's disease (FAD), termed Swedish mutant. Transfection of neuronal cells with HN cDNA or treatment with the coding HN polypeptide abrogated cytotoxicity by NL-APP. HN suppressed neurotoxicity by Abeta1-43 in the absence of N2 supplement, but could not inhibit Abeta secretion from NL-APP. HN could also protect neuronal cells from death by NL-APP lacking the 41st and 42nd residues of the Abeta region. Therefore, HN suppressed neuronal cell death by NL-APP not through inhibition of Abeta42 secretion, but with two targets for its inhibitory action: (i) the intracellular toxic mechanism directly triggered by NL-APP and (ii) neurotoxicity by Abeta. HN will contribute to the development of curative therapy of AD, especially as a novel reagent that could mechanistically supplement Abeta-production inhibitors.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Base Sequence; Cell Death; DNA, Complementary; Humans; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; Mutation; Neurons; Neuroprotective Agents; Proteins; Sweden; Transfection

Through functional expression screening, we identified a gene, designated Humanin (HN) cDNA, which encodes a short polypeptide and abolishes death of neuronal cells caused by multiple different types of familial Alzheimer's disease genes and by Abeta amyloid, without effect on death by Q79 or superoxide dismutase-1 mutants. Transfected HN cDNA was transcribed to the corresponding polypeptide and then was secreted into the cultured medium. The rescue action clearly depended on the primary structure of HN. This polypeptide would serve as a molecular clue for the development of new therapeutics for Alzheimer's disease targeting neuroprotection.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Protein Precursor; Cell Death; Cells, Cultured; Extracellular Space; Humans; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; Neurons; Peptides; Poly A; Proteins; Superoxide Dismutase; Superoxide Dismutase-1

A novel factor, termed Humanin (HN), antagonizes against neurotoxicity by various types of familial Alzheimer's disease (AD) genes [V642I and K595N/M596L (NL) mutants of amyloid precursor protein (APP), M146L-presenilin (PS) 1, and N141I-PS2] and by Abeta1-43 with clear action specificity ineffective on neurotoxicity by polyglutamine repeat Q79 or superoxide dismutase 1 mutants. Here we report that HN can also inhibit neurotoxicity by other AD-relevant insults: other familial AD genes (A617G-APP, L648P-APP, A246E-PS1, L286V-PS1, C410Y-PS1, and H163R-PS1), APP stimulation by anti-APP antibody, and other Abeta peptides (Abeta1-42 and Abeta25-35). The action specificity was further indicated by the finding that HN could not suppress neurotoxicity by glutamate or prion fragment. Against the AD-relevant insults, essential roles of Cys(8) and Ser(14) were commonly indicated, and the domain from Pro(3) to Pro(19) was responsible for the rescue action of HN, in which seven residues turned out to be essential. We also compared the neuroprotective action of S14G HN (HNG) with that of activity-dependent neurotrophic factor, IGF-I, or basic FGF for the antagonism against various AD-relevant insults (V642I-APP, NL-APP, M146L-PS1, N141I-PS2, and Abeta1-43). Although all of these factors could abolish neurotoxicity by Abeta1-43, only HNG could abolish cytotoxicities by all of them. HN and HN derivative peptides may provide a new insight into the study of AD pathophysiology and allow new avenues for the development of therapeutic interventions for various forms of AD.

Topics: Alzheimer Disease; Amino Acid Substitution; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Antibodies; Cell Death; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Fibroblast Growth Factor 2; Humans; Insulin-Like Growth Factor I; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Mice; Mutagenesis, Site-Directed; Mutation; Neurons; Neuroprotective Agents; Peptide Fragments; Presenilin-1; Proteins; Rats; Structure-Activity Relationship; Transfection