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.

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.

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.

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.

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.

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.

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.

Design and development of non-fibrillar amyloid beta as a potential Alzheimer vaccine.

Alzheimer's disease (AD) is the most common cause of dementia affecting the elderly. Treatment for effective cure of this complex neurodegenerative disease does not yet exist. In AD, otherwise soluble, monomeric form of amyloid beta (Abeta) peptide converts into toxic, fibrillar form rich in beta-sheet content. Several immunological approaches that prevent this conversion of Abeta into pathological form or that accelerate its clearance are being actively pursued worldwide. As part of these attempts, we report here, the design and characterization of a non-amyloidogenic homologue of Abeta (Abeta-KEK). We demonstrate that this peptide is helical in nature and retains the immunoneutralizing epitopes of native Abeta. More importantly, Fab fragments of the polyclonal anti-Abeta-KEK antibodies interfere with formation of Abeta fibrils as well as dissociate the preformed Abeta aggregates in vitro. These results suggest that non-amyloidogenic Abeta-KEK may serve as a safer alternative vaccine for Alzheimer's disease.

I32E and V36K double mutation in beta2-sheet abrogates amyloid beta peptide toxicity.

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, wherein, the accumulation of amyloid beta (Abeta) peptide as cytotoxic oligomers leads to neuropathologic changes. Transgenic mice with brain Abeta plaques immunized with aggregated Abeta have reduced amyloid burden and improved cognitive functions. However, such active immunization in humans led to a small but significant occurrence of meningoencephalitis in 6% AD volunteers due to Abeta induced toxicity. In an attempt to develop safer alternative vaccines, the design of a highly soluble peptide homologous to Abeta (Abeta-EK), that has a reduced amyloidogenic potential while maintaining the major immunogenic epitopes of Abeta is reported. More importantly, this homologue has been shown to be non-toxic, as this peptide failed to exert any observable effect on erythrocytes. The results of the present study suggests that immunization with non-toxic Abeta derivative may offer a safer therapeutic approach to AD, instead of using toxic Abeta fibrils.

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.

Influence of conformational antibodies on dissociation of fibrillar amyloid beta (A beta 1-42) in vitro.

Many neurodegenerative diseases result due to the accumulation of misfolded proteins as amyloid fibrils. Although the protein components of these fibrils from different disease states differ considerably, they appear to share common structure. Among these conformational disorders, Alzheimer's disease (AD) and prion diseases exhibit significant overlap in their mechanism of pathogenesis. The present report demonstrates that antibodies directed against the prion protein repeat motif, Tyr-Tyr-Arg motif, recognize recombinantly expressed human amyloid beta (A beta) aggregates in enzyme linked immunosorbent assay. In addition, these antibodies dissociate the preformed aggregates of A beta in vitro. These findings illustrate an important property of conformation dependent antibodies viz., they specifically recognize the protein deposits associated with pathology and not the protein in normal tissue. These antibodies may benefit the development of approaches towards prevention and treatment of protein misfolding diseases.

Enhanced Th2 immunity after DNA prime-protein boost immunization with amyloid beta (1-42) plus CpG oligodeoxynucleotides in aged rats.

Generation and accumulation of fibrillar amyloid beta (Abeta) is widely considered as the pathogenic basis of neurodegeneration in Alzheimer's disease (AD). Both active immunization with fibrillar Abeta and passive immunization with anti-Abeta antibodies in transgenic mouse models of AD result in prevention/dissociation of Abeta plaque formation and restoration of cognitive functions. However, similar immunization studies in humans had to be halted because 6% of the AD patients developed acute meningoencephalitis, likely due to anti-Abeta specific autoimmune Th1 cells. Hence, making Abeta immunotherapy successful requires production of strong antibody responses without Th1-type immunity. In an attempt to develop safer vaccines, we examined the influence of oligodeoxynucleotides as adjuvant on the Th1 and Th2 immune response to Abeta in aged rats. We further investigated whether a DNA prime-protein boost strategy could elicit a more robust Th2 response. The results of the present study showed that all the animals injected with either Abeta peptide alone or Abeta encoding plasmid alone or plasmid DNA prime followed by peptide boost have elicited specific anti-Abeta antibodies. When co-administered, synthetic oligodeoxynucleotides (ODN) further enhanced the anti-Abeta titres. More importantly, the IgG subclasses of the antibodies generated by DNA prime-peptide boost regimen with ODN as adjuvant were primarily of IgG2b and IgG1 isotypes, suggesting that heterologous immunization strategy along with ODN would be advantageous in eliciting more beneficial Th2-type humoral immune response.

Expression, purification and characterization of a synthetic gene encoding human amyloid beta (Abeta1-42) in Escherichia coli.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of cognitive function. Existing evidence indicates that abnormal processing and extracellular deposition of the longer form of the amyloid peptide Abeta(1-42), a proteolytic derivative of the amyloid precursor protein (APP), is a key step in the pathogenesis of AD. Active immunization with Abeta(1-42) has been shown to decrease brain beta deposition and improve cognitive performance in mouse models of AD. In the present study, we sought to express the synthetic gene encoding AB in Escherichia coli to enable rapid production of the antigen and its purification. The synthetic gene has been constructed from six oligonucleotides by employing overlapping PCR strategy and expressed in E. coli using the T7 promoter system. The recombinant peptide has been purified to homogeneity by a single step Ni+2 affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) using polyclonal anti-Abeta(1-42) sera confirms that the corresponding linear B-cell epitopic sequences are available for immunorecognition in the recombinant peptide. This methodology enables rapid, continuous production and purification in bulk amounts of human Abeta sequence by employing bacterial expression system

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.

Lower levels of cerebrospinal fluid amyloid beta (Abeta) in non-demented Indian controls.

Prevalence of Alzheimer's disease in Indian population is lower than in developed countries. To determine whether limitation of amyloid beta (Abeta) concentration may be responsible for lower rate of incidence, we measured the levels of Abeta in cerebrospinal fluid (CSF) collected from 72 non-demented individuals ranging in the age from 20 years to 65 years. These samples were segregated into three groups ranging from 20-35 years, 36-50 years and 51-65 years of age. Levels of Abeta could be detected in all the age groups and they were much lower than the values reported in literature from the developed countries. No significant difference in the average level of Ass was observed with increase in age.

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.