Antisense oligonucleotides targeting the miR-29b binding site in the GRN mRNA increase progranulin translation.

Heterozygous GRN (progranulin) mutations cause frontotemporal dementia (FTD) due to haploinsufficiency, and increasing progranulin levels is a major therapeutic goal. Several microRNAs, including miR-29b, negatively regulate progranulin protein levels. Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but strategies for increasing target protein levels are limited. Here, we tested the efficacy of ASOs as enhancers of progranulin expression by sterically blocking the miR-29b binding site in the 3' UTR of the human GRN mRNA. We found 16 ASOs that increase progranulin protein in a dose-dependent manner in neuroglioma cells. A subset of these ASOs also increased progranulin protein in iPSC-derived neurons and in a humanized GRN mouse model. In FRET-based assays, the ASOs effectively competed for miR-29b from binding to the GRN 3' UTR RNA. The ASOs increased levels of newly synthesized progranulin protein by increasing its translation, as revealed by polysome profiling. Together, our results demonstrate that ASOs can be used to effectively increase target protein levels by partially blocking miR binding sites. This ASO strategy may be therapeutically feasible for progranulin-deficient FTD as well as other conditions of haploinsufficiency.

Hepatocyte expression of the micropeptide adropin regulates the liver fasting response and is enhanced by caloric restriction.

The micropeptide adropin encoded by the clock-controlled energy homeostasis-associated gene is implicated in the regulation of glucose metabolism. However, its links to rhythms of nutrient intake, energy balance, and metabolic control remain poorly defined. Using surveys of Gene Expression Omnibus data sets, we confirm that fasting suppresses liver adropin expression in lean C57BL/6J (B6) mice. However, circadian rhythm data are inconsistent. In lean mice, caloric restriction (CR) induces bouts of compulsive binge feeding separated by prolonged fasting intervals, increasing NAD-dependent deacetylase sirtuin-1 signaling important for glucose and lipid metabolism regulation. CR up-regulates adropin expression and induces rhythms correlating with cellular stress-response pathways. Furthermore, adropin expression correlates positively with phosphoenolpyruvate carboxokinase-1 (Pck1) expression, suggesting a link with gluconeogenesis. Our previous data suggest that adropin suppresses gluconeogenesis in hepatocytes. Liver-specific adropin knockout (LAdrKO) mice exhibit increased glucose excursions following pyruvate injections, indicating increased gluconeogenesis. Gluconeogenesis is also increased in primary cultured hepatocytes derived from LAdrKO mice. Analysis of circulating insulin levels and liver expression of fasting-responsive cAMP-dependent protein kinase A (PKA) signaling pathways also suggests enhanced responses in LAdrKO mice during a glucagon tolerance test (250 µg/kg intraperitoneally). Fasting-associated changes in PKA signaling are attenuated in transgenic mice constitutively expressing adropin and in fasting mice treated acutely with adropin peptide. In summary, hepatic adropin expression is regulated by nutrient- and clock-dependent extrahepatic signals. CR induces pronounced postprandial peaks in hepatic adropin expression. Rhythms of hepatic adropin expression appear to link energy balance and cellular stress to the intracellular signal transduction pathways that drive the liver fasting response.

Adenosine A3 receptor as a novel therapeutic target to reduce secondary events and improve neurocognitive functions following traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is a common pathological condition that presently lacks a specific pharmacological treatment. Adenosine levels rise following TBI, which is thought to be neuroprotective against secondary brain injury. Evidence from stroke and inflammatory disease models suggests that adenosine signaling through the G protein-coupled A3 adenosine receptor (A3AR) can provide antiinflammatory and neuroprotective effects. However, the role of A3AR in TBI has not been investigated. METHODS: Using the selective A3AR agonist, MRS5980, we evaluated the effects of A3AR activation on the pathological outcomes and cognitive function in CD1 male mouse models of TBI. RESULTS: When measured 24 h after controlled cortical impact (CCI) TBI, male mice treated with intraperitoneal injections of MRS5980 (1 mg/kg) had reduced secondary tissue injury and brain infarction than vehicle-treated mice with TBI. These effects were associated with attenuated neuroinflammation marked by reduced activation of nuclear factor of kappa light polypeptide gene enhancer in B cells (NFκB) and MAPK (p38 and extracellular signal-regulated kinase (ERK)) pathways and downstream NOD-like receptor pyrin domain-containing 3 inflammasome activation. MRS5980 also attenuated TBI-induced CD4+ and CD8+ T cell influx. Moreover, when measured 4-5 weeks after closed head weight-drop TBI, male mice treated with MRS5980 (1 mg/kg) performed significantly better in novel object-placement retention tests (NOPRT) and T maze trials than untreated mice with TBI without altered locomotor activity or increased anxiety. CONCLUSION: Our results provide support for the beneficial effects of small molecule A3AR agonists to mitigate secondary tissue injury and cognitive impairment following TBI.

Biochemical, biomarker, and behavioral characterization of the GrnR493X mouse model of frontotemporal dementia.

Heterozygous loss-of-function mutations in the progranulin gene (GRN) are a major cause of frontotemporal dementia due to progranulin haploinsufficiency; complete deficiency of progranulin causes neuronal ceroid lipofuscinosis. Several progranulin-deficient mouse models have been generated, including both knockout mice and knockin mice harboring a common patient mutation (R493X). However, the GrnR493X mouse model has not been characterized completely. Additionally, while homozygous GrnR493X and Grn knockout mice have been extensively studied, data from heterozygous mice is still limited. Here, we performed more in-depth characterization of heterozygous and homozygous GrnR493X knockin mice, which includes biochemical assessments, behavioral studies, and analysis of fluid biomarkers. In the brains of homozygous GrnR493X mice, we found increased phosphorylated TDP-43 along with increased expression of lysosomal genes, markers of microgliosis and astrogliosis, pro-inflammatory cytokines, and complement factors. Heterozygous GrnR493X mice did not have increased TDP-43 phosphorylation but did exhibit limited increases in lysosomal and inflammatory gene expression. Behavioral studies found social and emotional deficits in GrnR493X mice that mirror those observed in Grn knockout mouse models, as well as impairment in memory and executive function. Overall, the GrnR493X knockin mouse model closely phenocopies Grn knockout models. Lastly, in contrast to homozygous knockin mice, heterozygous GrnR493X mice do not have elevated levels of fluid biomarkers previously identified in humans, including neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in both plasma and CSF. These results may help to inform pre-clinical studies that use this Grn knockin mouse model and other Grn knockout models.

Biochemical, Biomarker, and Behavioral Characterization of the GrnR493X Mouse Model of Frontotemporal Dementia.

Heterozygous loss-of-function mutations in the progranulin gene (GRN) are a major cause of frontotemporal dementia due to progranulin haploinsufficiency; complete deficiency of progranulin causes neuronal ceroid lipofuscinosis. Several progranulin-deficient mouse models have been generated, including both knockout mice and knockin mice harboring a common patient mutation (R493X). However, the GrnR493X mouse model has not been characterized completely. Additionally, while homozygous GrnR493X and Grn knockout mice have been extensively studied, data from heterozygous mice is still limited. Here, we performed more in-depth characterization of heterozygous and homozygous GrnR493X knockin mice, which includes biochemical assessments, behavioral studies, and analysis of fluid biomarkers. In the brains of homozygous GrnR493X mice, we found increased phosphorylated TDP-43 along with increased expression of lysosomal genes, markers of microgliosis and astrogliosis, pro-inflammatory cytokines, and complement factors. Heterozygous GrnR493X mice did not have increased TDP-43 phosphorylation but did exhibit limited increases in lysosomal and inflammatory gene expression. Behavioral studies found social and emotional deficits in GrnR493X mice that mirror those observed in Grn knockout mouse models, as well as impairment in memory and executive function. Overall, the GrnR493X knockin mouse model closely phenocopies Grn knockout models. Lastly, in contrast to homozygous knockin mice, heterozygous GrnR493X mice do not have elevated levels of fluid biomarkers previously identified in humans, including neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) in both plasma and CSF. These results may help to inform pre-clinical studies that use this Grn knockin mouse model and other Grn knockout models.

Targeting nonsense-mediated RNA decay does not increase progranulin levels in the Grn R493X mouse model of frontotemporal dementia.

A common cause of frontotemporal dementia (FTD) are nonsense mutations in the progranulin (GRN) gene. Because nonsense mutations activate the nonsense-mediated RNA decay (NMD) pathway, we sought to inhibit this RNA turnover pathway as a means to increase progranulin levels. Using a knock-in mouse model harboring a common patient mutation, we tested whether either pharmacological or genetic inhibition of NMD upregulates progranulin in these GrnR493X mice. We first examined antisense oligonucleotides (ASOs) targeting an exonic region in GrnR493X mRNA predicted to block its degradation by NMD. As we previously reported, these ASOs effectively increased GrnR493X mRNA levels in fibroblasts in vitro. However, following CNS delivery, we found that none of the 8 ASOs we tested increased Grn mRNA levels in the brains of GrnR493X mice. This result was obtained despite broad ASO distribution in the brain. An ASO targeting a different mRNA was effective when administered in parallel to wild-type mice. As an independent approach to inhibit NMD, we examined the effect of loss of an NMD factor not required for embryonic viability: UPF3b. We found that while Upf3b deletion effectively perturbed NMD, it did not increase Grn mRNA levels in Grn+/R493X mouse brains. Together, our results suggest that the NMD-inhibition approaches that we used are likely not viable for increasing progranulin levels in individuals with FTD caused by nonsense GRN mutations. Thus, alternative approaches should be pursued.

A DUAL MTOR/NAD+ ACTING GEROTHERAPY.

The geroscience hypothesis states that a therapy that prevents the underlying aging process should prevent multiple aging related diseases. The mTOR (mechanistic target of rapamycin)/insulin and NAD+ (nicotinamide adenine dinucleotide) pathways are two of the most validated aging pathways. Yet, it's largely unclear how they might talk to each other in aging. In genome-wide CRISPRa screening with a novel class of N-O-Methyl-propanamide-containing compounds we named BIOIO-1001, we identified lipid metabolism centering on SIRT3 as a point of intersection of the mTOR/insulin and NAD+ pathways. In vivo testing indicated that BIOIO-1001 reduced high fat, high sugar diet-induced metabolic derangements, inflammation, and fibrosis, each being characteristic of non-alcoholic steatohepatitis (NASH). An unbiased screen of patient datasets suggested a potential link between the anti-inflammatory and anti-fibrotic effects of BIOIO-1001 in NASH models to those in amyotrophic lateral sclerosis (ALS). Directed experiments subsequently determined that BIOIO-1001 was protective in both sporadic and familial ALS models. Both NASH and ALS have no treatments and suffer from a lack of convenient biomarkers to monitor therapeutic efficacy. A potential strength in considering BIOIO-1001 as a therapy is that the blood biomarker that it modulates, namely plasma triglycerides, can be conveniently used to screen patients for responders. More conceptually, to our knowledge BIOIO-1001 is a first therapy that fits the geroscience hypothesis by acting on multiple core aging pathways and that can alleviate multiple conditions after they have set in.

Pharmacokinetics and modeling of immune cell trafficking: quantifying differential influences of target tissues versus lymphocytes in SJL and lipopolysaccharide-treated mice.

BACKGROUND: Immune cell trafficking into the CNS and other tissues plays important roles in health and disease. Rapid quantitative methods are not available that could be used to study many of the dynamic aspects of immune cell-tissue interactions. METHODS: We used pharmacokinetics and modeling to quantify and characterize the trafficking of radioactively labeled lymphocytes into brain and peripheral tissues. We used variance from two-way ANOVAs with 2 × 2 experimental designs to model the relative influences of lymphocytes and target tissues in trafficking. RESULTS: We found that in male CD-1 mice, about 1 in 5,000 intravenously injected lymphocytes entered each gram of brain. Uptake by brain was 2 to 3 times higher in naïve SJL females, but uptake by spleen and clearance from blood was lower, demonstrating a dichotomy in immune cell distribution. Treatment of CD-1 mice with lipopolysaccharide (LPS) increased immune cell uptake into brain but decreased uptake by spleen and axillary nodes. CONCLUSIONS: Differences in brain uptake and in uptake by spleen between SJL and CD-1 mice were primarily determined by lymphocytes, whereas differences in uptake with LPS were primarily determined by lymphocytes for the brain but by the tissues for the spleen and the axillary lymph node. These results show that immune cells normally enter the CNS and that tissues and immune cells interact in ways that can be quantified by pharmacokinetic models.

Initial fate of prions upon peripheral infection: half-life, distribution, clearance, and tissue uptake.

Prion diseases are infectious neurodegenerative disorders associated with the misfolded prion protein (PrP(Sc)), which appears to be the sole component of the infectious agent (termed prion). To produce disease, prions have to be absorbed into the body and reach sufficient quantities in the brain. Very little is known about the biological mechanisms controlling the initial fate of prions. Here, we studied the systemic pharmacokinetics and biodistribution of PrP(Sc) in vivo. After an intravenous injection of highly purified radiolabeled or native unlabeled PrP(Sc), the protein was eliminated rapidly from the serum (half-life of 3.24 h), mostly through tissue uptake. The quantity of intact PrP(Sc) reaching the brain was ∼ 0.2% of the injected dose per gram of brain tissue (ID/g). The highest levels were found in liver (∼ 20% ID/g), spleen (∼ 13% ID/g), and kidney (∼ 7.4% ID/g). Cell surface PrP(C) does not appear to play a role in PrP(Sc) pharmacokinetics, since the infectious protein distributed similarly in wild-type and PrP-null mice. To measure tissue uptake kinetics and biodistribution accurately, vascular space in tissues was measured with radioactively labeled albumin coinjected with radioactively labeled PrP(Sc). Our results provide a fundamental pharmacokinetic characterization of PrP(Sc) in vivo, which may be relevant to estimate tissue risks and mechanisms of prion neuroinvasion and to identify novel therapeutic strategies.

Sphingosine-1-phosphate receptor 1 activation in the central nervous system drives cisplatin-induced cognitive impairment.

Cancer-related cognitive impairment (CRCI) is a major neurotoxicity affecting more than 50% of cancer survivors. The underpinning mechanisms are mostly unknown, and there are no FDA-approved interventions. Sphingolipidomic analysis of mouse prefrontal cortex and hippocampus, key sites of cognitive function, revealed that cisplatin increased levels of the potent signaling molecule sphingosine-1-phosphate (S1P) and led to cognitive impairment. At the biochemical level, S1P induced mitochondrial dysfunction, activation of NOD-, LRR-, and pyrin domain-containing protein 3 inflammasomes, and increased IL-1ß formation. These events were attenuated by systemic administration of the functional S1P receptor 1 (S1PR1) antagonist FTY720, which also attenuated cognitive impairment without adversely affecting locomotor activity. Similar attenuation was observed with ozanimod, another FDA-approved functional S1PR1 antagonist. Mice with astrocyte-specific deletion of S1pr1 lost their ability to respond to FTY720, implicating involvement of astrocytic S1PR1. Remarkably, our pharmacological and genetic approaches, coupled with computational modeling studies, revealed that cisplatin increased S1P production by activating TLR4. Collectively, our results identify the molecular mechanisms engaged by the S1P/S1PR1 axis in CRCI and establish S1PR1 antagonism as an approach to target CRCI with therapeutics that have fast-track clinical application.

Adropin transgenesis improves recognition memory in diet-induced obese LDLR-deficient C57BL/6J mice.

Obesity-related metabolic dysregulation causes mild cognitive impairment and increased risk for dementia. We used an LDLR-deficient C57BL/6J mouse model (LDLRKO) to investigate whether adropin, a neuropeptide linked to neurodegenerative diseases, improves cognitive function in situations of metabolic dysregulation. Adropin transgenic mice (AdrTG) were crossed with LDLRKO; male and female progeny were fed a high fat diet for 3-months. Male chow-fed wild type (WT) mice were used as controls. Diet-induced obesity and LDLR-deficiency caused severe dyslipidemia, irrespective of sex. The AdrTG prevented reduced adropin protein levels in LDLRKO cortex. In males, metabolic dysregulation and AdrTG genotype significantly and bi-directionally affected performance in the novel object recognition (NOR) test, a declarative hippocampal memory task (discrimination index mean ± SE for WT, 0.02 ± 0.088; LDLRKO, -0.115 ± 0.077; AdrTG;LDLRKO, 0.265 ± 0.078; genotype effect, p = 0.009; LDLRKO vs. AdrTG;LDLRKO, P < 0.05). A 2-way ANOVA (fixed variables: sex, AdrTG genotype) indicated a highly significant effect of AdrTG (P = 0.003). The impact of the diet-genotype interaction on the male mouse brain was investigated using RNA-seq. Gene-ontology analysis of transcripts showing fold-changes of>1.3 or <-1.3 (P < 0.05) indicated metabolic dysregulation affected gene networks involved in intercellular/neuronal signaling, immune processes, angiogenesis, and extracellular matrix organization. The AdrTG selectively attenuated the impact of metabolic dysregulation on intercellular/neuronal signaling pathways. Intercellular/neuronal signaling pathways were also the predominant processes overrepresented when directly comparing AdrTG;LDLRKO with LDRKO. In summary, adropin overexpression improves cognitive function in severe metabolic dysregulation through pathways related to cell-cell communication and neuronal processes, and independently of preventing inflammatory responses.

Synthesis and structure-activity relationships of 3,4,5-trisubstituted-1,2,4-triazoles: high affinity and selective somatostatin receptor-4 agonists for Alzheimer's disease treatment.

Somatostatin receptor-4 (SST4) is highly expressed in brain regions affiliated with learning and memory. SST4 agonist treatment may act to mitigate Alzheimer's disease (AD) pathology. An integrated approach to SST4 agonist lead optimization is presented herein. High affinity and selective agonists with biological efficacy were identified through iterative cycles of a structure-based design strategy encompassing computational methods, chemistry, and preclinical pharmacology. 1,2,4-Triazole derivatives of our previously reported hit (4) showed enhanced SST4 binding affinity, activity, and selectivity. Thirty-five compounds showed low nanomolar range SST4 binding affinity, 12 having a K i < 1 nM. These compounds showed >500-fold affinity for SST4 as compared to SST2A. SST4 activities were consistent with the respective SST4 binding affinities (EC50 < 10 nM for 34 compounds). Compound 208 (SST4 K i = 0.7 nM; EC50 = 2.5 nM; >600-fold selectivity over SST2A) display a favorable physiochemical profile, and was advanced to learning and memory behavior evaluations in the senescence accelerated mouse-prone 8 model of AD-related cognitive decline. Chronic administration enhanced learning with i.p. dosing (1 mg kg-1) compared to vehicle. Chronic administration enhanced memory with both i.p. (0.01, 0.1, 1 mg kg-1) and oral (0.01, 10 mg kg-1) dosing compared to vehicle. This study identified a novel series of SST4 agonists with high affinity, selectivity, and biological activity that may be useful in the treatment of AD.

Adropin correlates with aging-related neuropathology in humans and improves cognitive function in aging mice.

The neural functions of adropin, a secreted peptide highly expressed in the brain, have not been investigated. In humans, adropin is highly expressed in astrocytes and peaks during critical postnatal periods of brain development. Gene enrichment analysis of transcripts correlating with adropin expression suggests processes relevant to aging-related neurodegenerative diseases that vary with age and dementia state, possibly indicating survivor bias. In people aged <40 y and 'old-old' (>75 y) diagnosed with dementia, adropin correlates positively with genes involved in mitochondrial processes. In the 'old-old' without dementia adropin expression correlates positively with morphogenesis and synapse function. Potent neurotrophic responses in primary cultured neurons are consistent with adropin supporting the development and function of neural networks. Adropin expression in the 'old-old' also correlates positively with protein markers of tau-related neuropathologies and inflammation, particularly in those without dementia. How variation in brain adropin expression affects neurological aging was investigated using old (18-month) C57BL/6J mice. In mice adropin is expressed in neurons, oligodendrocyte progenitor cells, oligodendrocytes, and microglia and shows correlative relationships with groups of genes involved in neurodegeneration and cellular metabolism. Increasing adropin expression using transgenesis improved spatial learning and memory, novel object recognition, resilience to exposure to new environments, and reduced mRNA markers of inflammation in old mice. Treatment with synthetic adropin peptide also reversed age-related declines in cognitive functions and affected expression of genes involved in morphogenesis and cellular metabolism. Collectively, these results establish a link between adropin expression and neural energy metabolism and indicate a potential therapy against neurological aging.

A pharmacologically active monoclonal antibody against the human melanocortin-4 receptor: effectiveness after peripheral and central administration.

The hypothalamic melanocortin-4 receptor (MC4R) is a constituent of an important pathway regulating food intake and energy expenditure. We produced a monoclonal antibody (mAb) directed against the N-terminal domain of the MC4R and evaluated its potential as a possible therapeutic agent. This mAb (1E8a) showed specific binding to the MC4R in human embryonic kidney 293 cells expressing the human MC4R and blocked the activity of the MC4R under basal conditions and after stimulation with alpha-melanocyte-stimulating hormone (alpha-MSH). The inverse agonist action of Agouti-related protein was significantly enhanced in the presence of mAb 1E8a. After a single intracerebroventricular injection into the third ventricle, mAb 1E8a (1 microg) increased 24-h food intake in rats. After 7 days of continuous intracerebroventricular administration, mAb 1E8a increased food intake, body weight, and fat pad weight and induced hyperglycemia. Because the complete mAb was ineffective after intravenous injection, we produced single-chain variable fragments (scFvs) derived from mAb 1E8a. In pharmacokinetic studies it was demonstrated that these scFvs crossed the blood-brain barrier and reached the hypothalamus. Consequently, the scFv 1E8a increased significantly food intake and body weight in rats after intravenous administration (300 mug/kg). The pharmacological profile of mAb 1E8a and the fact that its scFv was active after peripheral administration suggest that derivatives of anti-MC4R mAbs may be useful in the treatment of patients with anorexia or cachexia.

Serum progranulin levels are associated with frailty in middle-aged individuals.

BACKGROUND AND OBJECTIVE: A recent study identified progranulin as a candidate biomarker for frailty, based on gene expression databases. In the present study, we investigated associations between serum progranulin levels and frailty in a population-based sample of late middle-age and older adults. METHODS: We utilized a cohort study that included 358 African Americans (baseline ages 49-65). Frailty was assessed by three established methods: the interview-based FRAIL scale, the Cardiovascular Health Study (CHS) frailty scale that includes performance-based measurements, and the Frailty Index (FI) that is based on cumulative deficits. Serum levels of the following proteins and metabolites were measured: progranulin, cystatin C, fructosamine, soluble cytokine receptors (interleukin-2 and -6, tumor necrosis factor α-1 and -2), and C-reactive protein. Sarcopenia was assessed using the SARC-F index. Vital status was determined by matching through the National Death Index (NDI). RESULTS: Serum progranulin levels were associated with frailty for all indices (FRAIL, CHS, and FI) but not with sarcopenia. Inflammatory markers indicated by soluble cytokine receptors (sIL-2R, sIL-6R, sTNFR1, sTNFR2) were positively associated serum progranulin. Increased serum progranulin levels at baseline predicted poorer outcomes including future frailty as measured by the FRAIL scale and 15-year all-cause mortality independent of age, gender, and frailty. CONCLUSIONS: Our findings suggest that serum progranulin levels may be a candidate biomarker for physical frailty, independent of sarcopenia. Further studies are needed to validate this association and assess the utility of serum progranulin levels as a potential biomarker for prevalent frailty, for risk for developing incident frailty, and for mortality risk over and above the effect of baseline frailty.

Metformin Improves Learning and Memory in the SAMP8 Mouse Model of Alzheimer's Disease.

Metformin is used for the treatment of insulin resistant diabetes. Diabetics are at an increased risk of developing dementia. Recent epidemiological studies suggest that metformin treatment prevents cognitive decline in diabetics. A pilot clinical study found cognitive improvement with metformin in patients with mild cognitive impairment (MCI). Preclinical studies suggest metformin reduces Alzheimer-like pathology in mouse models of Alzheimer's disease (AD). In the current study, we used 11-month-old SAMP8 mice. Mice were given daily injections of metformin at 20 mg/kg/sc or 200 mg/kg/sc for eight weeks. After four weeks, mice were tested in T-maze footshock avoidance, object recognition, and Barnes maze. At the end of the study, brain tissue was collected for analysis of PKC (PKCζ, PKCι, PKCα, PKCγ, PKCɛ), GSK-3ß, pGSK-3ßser9, pGSK-3ßtyr216, pTau404, and APP. Metformin improved both acquisition and retention in SAMP8 mice in T-maze footshock avoidance, retention in novel object recognition, and acquisition in the Barnes maze. Biochemical analysis indicated that metformin increased both atypical and conventional forms of PKC; PKCζ, and PKCα at 20 mg/kg. Metformin significantly increased pGSK-3ßser9 at 200 mg/kg, and decreased Aß at 20 mg/kg and pTau404 and APPc99 at both 20 mg/kg and 200 mg/kg. There were no differences in blood glucose levels between the aged vehicle and metformin treated mice. Metformin improved learning and memory in the SAMP8 mouse model of spontaneous onset AD. Biochemical analysis indicates that metformin improved memory by decreasing APPc99 and pTau. The current study lends support to the therapeutic potential of metformin for AD.

Inhibition of Glycogen Synthase Kinase 3ß as a Treatment for the Prevention of Cognitive Deficits after a Traumatic Brain Injury.

Traumatic brain injury (TBI) has many long-term consequences, including impairment in memory and changes in mood. Glycogen synthase kinase 3ß (GSK-3ß) in its phosphorylated form (p-GSK-3ß) is considered to be a major contributor to memory problems that occur post-TBI. We have developed an antisense that targets the GSK-3ß (GAO) gene. Using a model of closed-head concussive TBI, we subjected mice to TBI and injected GAO or a random antisense (RAO) 15 min post-injury. One week post-injury, mice were tested in object recognition with 24 h delay. At 4 weeks post- injury, mice were tested with a T-maze foot shock avoidance memory test and a second object recognition test with 24 h delay using different objects. Mice that received GAO show improved memory in both object recognition and T-maze compared with RAO- treated mice that were subjected to TBI. Next, we verified that GAO blocked the surge in phosphorylated GSK-3ß post-TBI. Mice were subjected to TBI and injected with antisense 15 min post-TBI with GAO or RAO. Mice were euthanized at 4 and 72 h post-TBI. Analysis of p-ser9GSK-3ß, p-tyr216GSK-3ß, and phospho-tau (p-tau)404 showed that mice that received a TBI+RAO had significantly higher p-ser9GSK-3ß, p-tyr216GSK-3ß, and p-tau404 levels than the mice that received TBI+GAO and the Sham+RAO mice. The current finding suggests that inhibiting GSK-3ß increase after TBI with an antisense directed at GSK-3ß prevents learning and memory impairments.

Molecular Mechanisms of Intranasal Insulin in SAMP8 Mice.

Research on intranasal delivery of drugs, peptides, and proteins has grown over the past decade as an alternate way to deliver substrates to the brain. Recent work has shown intranasal (INL) delivery of insulin improves memory and cognition in healthy subjects as well as patients with Alzheimer's disease (AD) and in AD mouse models. However, the molecular mechanism(s) for the beneficial effect of insulin on memory are still unclear. Using the SAMP8 mouse model of AD, we investigated the impact of INL insulin on protein and gene expression in brain regions including the olfactory bulb, hypothalamus, and hippocampus. We found genes and proteins in the insulin receptor signaling pathway were not activated by the doses tested. However, we did find the expression of genes present in the hippocampus involved in other pathways, especially those related to inflammation, were altered due to age and with a dose of INL insulin previously shown to improve cognition. These alternate pathways could be targets of insulin when delivered via the INL route to aid in memory improvement.

Permeability of the blood-brain barrier to a novel satiety molecule nesfatin-1.

Nesfatin-1 has recently been identified as a hypothalamic and brain stem peptide that regulates feeding behavior. Here, we determined the ability of nesfatin-1 to cross the blood-brain barrier (BBB) of mice. We used multiple-regression analysis to determine that radioactively labeled nesfatin-1 injected intravenously entered the brain. The entry rate (K(i)) of (131)I-nesfatin-1 from blood-to-brain was 0.20+/-0.02 microl/g min. This modest rate of entry was not inhibited by the administration of nonradioactive nesfatin-1, suggesting that BBB transport of nesfatin-1 into the brain is by a nonsaturable mechanism. High performance liquid chromatography (HPLC) and acid precipitation showed that most of the injected radiolabeled nesfatin-1 reached the brain as intact peptide, and capillary depletion with vascular washout revealed that 67% of (131)I-nesfatin-1 crossed the BBB to reach the brain parenchyma. Efflux of labeled nesfatin-1 from brain back into blood was by way of bulk flow. These findings demonstrate that nesfatin-1 crosses the BBB in both the blood-to-brain and brain-to-blood directions by nonsaturable mechanisms.

Aluminum complexing enhances amyloid beta protein penetration of blood-brain barrier.

A significant co-morbidity of Alzheimer's disease and cerebrovascular impairment suggests that cerebrovascular dysregulation is an important feature of dementia. Amyloid beta protein (Abeta), a relevant risk factor in Alzheimer's disease, has neurotoxic properties and is thought to play a critical role in the cognitive impairments. Previously, we demonstrated that the 42mer of Abeta (Abeta42) complexed with aluminum (Al-Abeta42) is much more cytotoxic than non-complexed Abeta42. The level of Abeta in the brain is a balance between synthesis, degradation, and fluxes across the blood-brain barrier (BBB). In the present paper, we determined whether complexing with aluminum affected the ability of radioactively iodinated Abeta to cross the in vivo BBB. We found that the rates of uptake of Al-Abeta42 and Abeta42 were similar, but that Al-Abeta42 was sequestered by brain endothelial cells much less than Abeta42 and so more readily entered the parenchymal space of the brain. Al-Abeta42 also had a longer half-life in blood and had increased permeation at the striatum and thalamus. Brain-to-blood transport was similar for Al-Abeta42 and Abeta42. In conclusion, complexing with aluminum affects some aspects of blood-to-brain permeability so that Al-Abeta42 would have more ready access to brain cells than Abeta42.