Potential Utility of Plasma Biomarker Panels in Differential Diagnosis of Alzheimer's Disease.

Blood tests are in need, in the clinical diagnosis of Alzheimer's disease (AD) as minimally invasive and less expensive alternatives to cerebrospinal fluid and neuroimaging methods. On these lines, single molecule array (Simoa) analysis of amyloid-ß (Aß42), total tau (t-tau), phospho-tau (p-tau 181), and neurofilament L (NfL) in the plasma samples of AD subjects, healthy controls (HC), and non-AD subjects was conducted. Findings from this study suggest that a panel of multiple plasma biomarkers (NfL, Aß42, t-tau, and p-tau 181) combined with the clinical assessments could support differential diagnosis of AD and other dementias from healthy controls.

Pathological (Dis)Similarities in Neuronal Exosome-Derived Synaptic and Organellar Marker Levels Between Alzheimer's Disease and Frontotemporal Dementia.

BACKGROUND: Alzheimer's disease (AD) and frontotemporal dementia (FTD) are pathologically distinct neurodegenerative disorders with certain overlap in cognitive and behavioral symptoms. Both AD and FTD are characterized by synaptic loss and accumulation of misfolded proteins, albeit, in different regions of the brain. OBJECTIVE: To investigate the synaptic and organellar markers in AD and FTD through assessment of the levels of synaptic protein, neurogranin (Ng) and organellar proteins, mitofusin-2 (MFN-2), lysosomal associated membrane protein-2 (LAMP-2), and golgin A4 from neuronal exosomes. METHODS: Exosomes isolated from the plasma of healthy controls (HC), AD and FTD subjects were characterized using transmission electron microscopy. Neurodegenerative status was assessed by measurement of neurofilament light chain (NfL) using Simoa. The pooled exosomal extracts from each group were analyzed for Ng, MFN-2, LAMP-2, and golgin A4 by western blot analysis using enhanced chemiluminescence method of detection. RESULTS: The densitometric analysis of immunoreactive bands demonstrated a 65% reduction of Ng in AD and 53% in FTD. Mitochondrial protein MFN-2 showed a significant reduction by 32% in AD and 46% in FTD. Lysosomal LAMP-2 and Golgi complex associated golgin A4 were considerably increased in both AD and FTD. CONCLUSION: Changes in Ng may reflect the ongoing synaptic degeneration that are linked to cognitive disturbances in AD and FTD. Importantly, the rate of synaptic degeneration was more pronounced in AD. Changes to a similar extent in both the dementia groups in organellar proteins indicates shared mechanisms of protein accumulation/degradation common to both AD and FTD.

Plasma neurofilament L to amyloid ß42 ratio in differentiating Alzheimer's type from non-Alzheimer's dementia: A cross-sectional pilot study from India.

Based on the reduction of amyloid ß plaques, US FDA has recently approved Aducanumab as a disease modifying treatment for Alzheimer's disease (AD). With high pricing and the potential risks likely with this treatment, certainty of AD diagnosis becomes crucial. The current pilot study evaluated plasma levels of neurofilament L, an axonal injury marker and amyloid ß42, a major component of amyloid plaques for discriminating AD from non-AD dementia (NAD). Results with Simoa assays indicate that a combination of neurofilament L and amyloid ß42 can be considered as a screening tool in identifying eligible subjects for AD treatment/ clinical trials.

Increased prolidase activity in Alzheimer's dementia: A case-control study.

Prolidase enzyme, which catalyzes the final step in collagen metabolism can influence the cognitive functions through changes in extracellular matrix (ECM) resulting in altered synaptic connectivity in Alzheimer's disease (AD). In this study, it was found that the prolidase activity was significantly higher (p = 0.0016) in AD subjects (5.62 ± 2.05 U/ mL) than control group (4.45 ± 0.92 U/ mL). The increase was significant beginning at mild AD (p = 0.006) with an inverse correlation with HMSE scores (p = 0.0344), thus implying that prolidase mediated alterations in ECM may be associated with the cognitive deficits seen in AD.

Elevated serum adenosine deaminase levels in neuroleptic-naïve patients with recent-onset schizophrenia.

The present study examined serum levels of adenosine deaminase (ADA), an adenosine metabolizing enzyme, in neuroleptic-naive patients with recent-onset schizophrenia and age-matched healthy comparison subjects. ADA levels were found to be higher among patients, and revealed a possible link between evening rise and severity of auditory hallucinations as well as morning rise and severity of avolition-apathy in patients with schizophrenia. These findings suggest the potential utility of serum ADA as a peripheral biomarker of schizophrenia.

Passive immunization targeting the N-terminal region of phosphorylated tau (residues 68-71) improves spatial memory in okadaic acid induced tauopathy model rats.

Alzheimer's disease (AD) is the leading cause of dementia, characterized by progressive loss of memory and other cognitive functions. The cognitive impairment in patients with AD is closely associated with loss of synapses and the formation of neurofibrillary tangles (NFT) containing hyperphosphorylated tau in the hippocampus. Effective treatment for AD is still not available. In this study, the sequence comprising of residues 50-71 in the N-terminal region of tau, containing theoretically predicted B- and T-cell epitopes in close proximity to pathologically relevant phospho-serine (residue 68) and phospho-threonine (residues 69, 71) was selected as a potential immunotherapeutic peptide. This 22-residue long phospho-peptide (50TPTEDGSEEPGSETSDAKpSpTPpT71) was custom synthesized and its therapeutic potential was tested in experimental rats. For this purpose, adult Sprague-Dawley rats were intranasally treated with okadaic acid (OA), a selective inhibitor of protein phosphatase PP2A. Within a day of OA administration, these rats showed marked impairment in cognitive functions with a significant increase in p-tau/t-tau ratio in the hippocampal homogenates. Passive immunization studies conducted in these OA treated rats with polyclonal anti-phospho-peptide antibodies resulted in a significant improvement in learning and memory functions in Barne's maze task. Further, p-tau levels in the hippocampal homogenates were reduced. In addition, these antibodies effectively prevented the aggregation of recombinant tau in vitro. These results demonstrate that targeting N-terminal region of tau harbouring the phospho-residue cluster 68-71 would be beneficial and may present an effective therapeutic opportunity for AD and other tauopathies.

Cognition enhancing effect of the aqueous extract of Cinnamomum zeylanicum on non-transgenic Alzheimer's disease rat model: Biochemical, histological, and behavioural studies.

OBJECTIVE: Several dietary supplements are actively being tested for their dual role of alleviating the metabolic perturbations and restricting the consequent cognitive dysfunctions seen in neurodegenerative disorders such as Alzheimer's disease (AD). The aim of the current study was to assess the influence of aqueous extract of cinnamon (CE) on the monosodium glutamate-induced non-transgenic rat model of AD (NTAD) established with insulin resistance, hyperglycaemia, neuronal loss, and cognitive impairment at a very early stage of life. METHODS: The experimental design included oral administration of CE (50 mg/kg body weight) for 20 weeks to 2-month and 10-month-old NTAD rats. Following the treatments, the animals attained 7 and 15 months of age, respectively. They were then subjected to behavioural testing, biochemical analysis, and stereology experiments. RESULTS: The results demonstrated that CE treatment improved the insulin sensitivity, increased phosphorylated glycogen synthase kinase-3ß (pGSK3ß), inhibited the cholinesterase activity, and improved the learning ability in NTAD rats. Histological evaluation has shown an increase in neuron count in the DG sub-field of hippocampus upon treatment with CE. DISCUSSION: These beneficial effects of CE are suggestive of considering cinnamon as a dietary supplement in modulating the metabolic changes and cognitive functions.

Development of a dot blot assay with antibodies to recombinant "core" 14-3-3 protein: Evaluation of its usefulness in diagnosis of Creutzfeldt-Jakob disease.

BACKGROUND AND PURPOSE: Definitive diagnosis of Creutzfeldt-Jakob disease (CJD) requires demonstration of infective prion protein (PrP(Sc)) in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO) recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF) in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy. This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD. MATERIALS AND METHODS: A minigene expressing the "core" 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR) and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF. RESULTS: The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001) in the CSF levels of 14-3-3 protein between the CJD cases (N= 50) and disease controls (N= 70). The receiver operating characteristic (ROC) analysis of the results suggested an optimal cutoff value of 2 ng/mL. CONCLUSIONS: We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF.

The Neuroprotective Effect of Dark Chocolate in Monosodium Glutamate-Induced Nontransgenic Alzheimer Disease Model Rats: Biochemical, Behavioral, and Histological Studies.

The vulnerability to oxidative stress and cognitive decline continue to increase during both normal and pathological aging. Dietary changes and sedentary life style resulting in mid-life obesity and type 2 diabetes, if left uncorrected, further add to the risk of cognitive decline and Alzheimer disease (AD) in the later stages of life. Certain antioxidant agents such as dietary polyphenols, taken in adequate quantities, have been suggested to improve the cognitive processes. In this study, we examined the effect of oral administration of dark chocolate (DC) containing 70% cocoa solids and 4% total polyphenol content for three months at a dose of 500 mg/Kg body weight per day to 17-month-old monosodium glutamate treated obese Sprague-Dawley rats, earlier characterized as a nontransgenic AD (NTAD) rat model after reversal of obesity, diabetes, and consequent cognitive impairments. The results demonstrated that DC reduced the hyperglycemia, inhibited the cholinesterase activity in the hippocampal tissue homogenates, and improved the cognitive performance in spatial memory related Barnes maze task. Histological studies revealed an increase in cell volume in the DC treated rats in the CA3 region of the hippocampus. These findings demonstrated the benefits of DC in enhancing cognitive function and cholinergic activity in the hippocampus of the aged NTAD rats while correcting their metabolic disturbances.

Combination of Spirulina with glycyrrhizin prevents cognitive dysfunction in aged obese rats.

OBJECTIVES: To evaluate the cognition enhancing effect of the combination of Spirulina and glycyrrhizin in monosodium glutamate (MSG)-induced obese aged rats. MATERIALS AND METHODS: Obesity was induced in rats by administration of MSG (intraperitoneally, 4 mg/g body weight) for 14 consecutive days from day 1 after birth. Subsequently, the animals were allowed to grow for 18 months with food and water ad libitum. Hypercholesterolemia, hyperglycemia, leptin resistance, were monitored in these animals. Cognitive status was assessed by Barne's maze task and hippocampal acetylcholinesterase (AChE) levels. Further, the animals were treated with Spirulina (Sp) (oral route, 1 g/Kg body weight, for 30 days) alone or glycyrrhizin (Gly) alone (intraperitoneal route, 0.1 mg/Kg, on day 15 and day 21), or their combination (SpGly). Counting of the treatment days was done by considering first day of Sp administration as day 1. After the completion of 30 days of Spirulina treatment or 2 doses of Gly administration or the combination (SpGly) treatment, the animals were left for 3 weeks. They were then were assessed for their biochemical and cognitive changes. RESULTS: The combination of Sp with Gly showed a significant reduction (P < 0.0001) in glucose, cholesterol, leptin levels in the serum with improvement in cognitive functions with concomitant reduction in AChE activity in the hippocampal tissue homogenates (P < 0.0001) of the obese rats. CONCLUSION: SpGly combination has a potential role in reversing cognitive dysfunctions associated with aging and obesity.

Supportive CSF biomarker evidence to enhance the National Institute on Aging-Alzheimer's Association criteria for diagnosis of Alzheimer's type dementia--a study from Southern India.

The present study was undertaken to validate the measurement of biomarkers as a supplement to the latest diagnostic criteria for Alzheimer disease (AD) dementia by National Institute on Aging-Alzheimer's Association (NIA-AA) work group using a sample attending a tertiary care center in Southern India. A total of 20 subjects diagnosed clinically as Alzheimer's dementia according to the NIA-AA criteria for AD were included in the study. The CSF biomarkers Aß42, t-tau, and p-tau181 were assessed. The biomarker results were compared among mild and moderate to severe AD as defined in the NIA-AA work group guidelines. The results revealed that the amount of Aß42 was very low in all the 20 samples (<50pg/ml) collected from mild AD cases with CDR score of 1 (n=8), and moderate to severe AD cases with CDR >1 (n=12). t-tau and p-tau levels were in the range of 39.45±5.09pg/ml and 13.06±7.32pg/ml for CDR 1 group. t-tau and p-tau levels were in the range of 49.9±11.28pg/ml and 33.94±15.13pg/ml for moderate to severe cases. Analysis of the data revealed statistically significant differences in the p-tau/t-tau ratio and p-tau/Aß ratio between CDR 1and CDR >1 AD cases (p<0.001) suggesting that p-tau/t-tau and p-tau/Aß ratio are good indicators of severity of dementia with discriminative value in differentiating mild AD from moderate to severe AD.

Amyloid beta lowering and cognition enhancing effects of ghrelin receptor analog [D-Lys (3)] GHRP-6 in rat model of obesity.

Obesity arising due to the dietary and life style changes is fast reaching epidemic proportions all over the world. There is increasing evidence that the incidence of Alzheimer disease (AD) is significantly influenced by a cluster of metabolic diseases, including diabetes and obesity. This study was aimed to test the suitability of experimentally-induced obesity in rats as an experimental animal model of AD. We used the procedure of neonatal administration of rats with monosodium L-glutamate (MSG), which generates adult obese animals as our study design and assessed the AD-like changes by measuring amyloid beta (1-42) and acetylcholinesterase (AChE) levels in the hippocampal extracts and cognitive impairments by Barnes maze task. Further, we investigated the influence of anti-obesity substance [D-Lys (3)] GHRP-6 on blood glucose, hippocampal Abeta, AChE levels and restoration of cognitive deficits. Results revealed that administration of MSG to neonatal rats exhibited increased body mass index and serum glucose levels over the controls. Measurement of markers for AD-like molecular changes i.e. amyloid beta (Abeta) and AChE levels showed marked elevation in these two parameters in the hippocampus of MSG-treated rats. Assessment of cognitive abilities by Barnes maze revealed spatial disorientation characteristic of AD. Administration of ghrelin receptor analog [D-Lys (3)] GHRP-6 to obese rats resulted in significant restoration of serum cholesterol, glucose, leptin and ghrelin levels to that of control with concomitant reduction in hippocampal Abeta and AChE levels. In addition, the treated animals exhibited marked improvement in Barne's maze task. These findings suggest that MSG-induced obese rats may serve as non-transgenic animal model for AD research. Further, the results indicate the potential of [D-Lys (3)] GHRP-6 as a promising anti-Alzheimer candidate.

In vitro evaluation of anti-Alzheimer effects of dry ginger (Zingiber officinale Roscoe) extract.

As the disease modifying therapies against Alzheimer's disease (AD) continue to exist as a major challenge of this century, the search for newer drug leads with lesser side effects is on the rise. A large number of plant extracts and phytocompounds are being actively pursued for their anti-Alzheimer effects. In the present study, the antioxidant activity, cholinesterase inhibition, anti-amyloidogenic potential and neuroprotective properties of methanolic extract of dry ginger (GE) have been evaluated. The extract contained 18 +/- 0.6 mg/g gallic acid equivalents of total phenolic content and 4.18 +/- 0.69 mg quercetin equivalents/g of dry material. GE expressed high antioxidant activity with an IC50 value of 70 +/- 0.304 microg/mL in DPPH assay and 845.4 +/- 56.62 microM Fe(II) equivalents/g dry weight in FRAP assay respectively. In Ellman's assay for the cholinesterase inhibitory activity, GE had an IC50 value of 41 +/- 1.2 microg/mL and 52 +/- 2 microg/mL for inhibition of acetyl- and butyrylcholinesterase respectively. Also, GE increased the cell survival against amyloid beta (Abeta) induced toxicity in primary adult rat hippocampal cell culture. Aggregation experiments with the thioflavin T binding studies showed that GE effectively prevented the formation of Abeta oligomers and dissociated the preformed oligomers. These findings suggest that methanolic GE influences multiple therapeutic molecular targets of AD and can be considered as an effective nontoxic neutraceutical supplement for AD.

In vitro screening for anti-cholinesterase and antioxidant activity of methanolic extracts of ayurvedic medicinal plants used for cognitive disorders.

Inhibition of Acetylcholinesterase (AChE) is still considered as the main therapeutic strategy against Alzheimer's disease (AD). Many plant derived phytochemicals have shown AChE inhibitory activity in addition to the currently approved drugs for AD. In the present study, methanolic extracts of 20 plants used in Indian Ayurvedic system of medicine for improving cognitive function were screened for acetylcholinesterase inhibitory activity by Ellman's microplate colorimetric method. Out of 20 extracts, Emblica officinalis, Nardostachys jatamansi, Nelumbo nucifera, Punica granatum and Raulfia Serpentina showed IC50 values <100 µg/ml for acetylcholinesterase inhibitory activity. Antioxidant activities of these plants were assessed by DPPH scavenging assay. Among the extracts used, antioxidant activity was highest for Terminalia chebula and Emblica officinalis with IC50 values <10 µg/ml. Considering the complex multifactorial etiology of AD, these plant extracts will be safer and better candidates for the future disease modifying therapies against this devastating disease.

Intranasal administration of insulin lowers amyloid-beta levels in rat model of diabetes.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by abnormal accumulation of amyloid beta (A beta) peptide in brain regions subserving memory and other cognitive functions. Hyperglycemia and perturbed insulin signaling have been proposed as pathogenic factors contributing to AD. The aim of the present study is to validate the use of streptozotocin (STZ) injected rats as an experimental model of AD. Using this model, the effect of intranasal administration of insulin on reduction of A beta levels was measured. The current findings strengthen the case for insulin as therapy for AD afflicted individuals with or without diabetes.

Identification and mapping of linear antigenic determinants of human amyloid ß(1-42) peptide.

Accumulation of cytotoxic oligomers of amyloid ß (Aß) is one of the major pathological hallmarks of Alzheimer's disease (AD). Several immunological approaches that prevent the conversion of Aß into its toxic form or that accelerate its clearance are being actively pursued worldwide. As part of these attempts, we have carried out sequential epitope analysis of Aß where antibodies raised against native Aß and its homologue Aß-KEK were screened for binding to five overlapping hexadecapeptides encompassing the full length of Aß sequence with 10 amino acid overlap. By this approach, we could identify a neutralizing epitope spanning the region 13-28 in Aß. These results demonstrate the presence of an additional stretch of Aß that can serve as mini-vaccine for AD.