Two behavioral tests allow a better correlation between cognitive function and expression of synaptic proteins

The molecular substrate of age-associated cognitive decline (AACD) is still elusive. Evidence indicates that AACD is related to synaptic impairment in hippocampus, but different hippocampal regions play different roles, with the dorsal hippocampus (DH) associated to spatial learning, and the ventral hippocampus (VH) crucial for emotionality. If changes in hippocampal function contributes to AACD, this contribution may be reflected in alterations of synaptic protein levels. A commonly used approach to investigate this issue is western blotting. When this technique is applied to the entire hippocampus and the cognitive impairment is evaluated by a single task, changes in expression of a protein might undergo a "dilution effect", as they may occur only in a given hippocampal region. We show that two behavioral tests yield more accurate results than one test in evaluating the function of the whole rat hippocampus by studying the expression of synaptotagmin 1 (SYT1), a vesicular protein whose expression in aged hippocampus is reportedly inconsistent. Analysis of SYT1 levels in the whole hippocampus of rats selected by the Morris water maze (MWM) test only failed to highlight a difference, whereas analysis of SYT1 levels in the whole hippocampus of rats categorized by both the MWM and the step-through passive avoidance (STPA) tests demonstrated a significant increase of SYT1 level in impaired rats. These findings, besides showing that SYT1 increases in impaired aged rats, suggest that using the whole hippocampus in blotting studies may prevent false negative results only if animals are categorized with tests exploring both DH and VH

Morphological and functional study of cerebral aging in rat

Aging is a physiological process characterized by a progressive decline of the “cellular homeostatic reserve”, refereed as the capability to respond suitably to exogenous and endogenous stressful stimuli. Due to their high energetic requests and post-mitotic nature, neurons are peculiarly susceptible to this phenomenon. However, the aged brain maintains a certain level of adaptive capacities and if properly stimulated may warrant a considerable functional recovery. Aim of the present research was to verify the plastic potentialities of the aging brain of rats subjected to two kind of exogenous stimuli: A) the replacement of the standard diet with a ketogenic regimen (the change forces the brain to use ketone bodies (KB) in alternative to glucose to satisfy the energetic needs) and B) a behavioural task able to induce the formation of inhibitory avoidance memory. A) Fifteen male Wistar rats of 19 months of age were divided into three groups (average body weight pair-matched), and fed for 8 weeks with different dietary regimens: i) diet containing 10% medium chain triglycerides (MCT); ii) diet containing 20% MCT; iii) standard commercial chow. Five young (5 months of age) and five old (26-27 months of age) animals fed with the standard diet were used as further controls. The following morphological parameters reflecting synaptic plasticity were evaluated in the stratum moleculare of the hippocampal CA1 region (SM CA1), in the outer molecular layer of the hippocampal dentate gyrus (OML DG), and in the granule cell layer of the cerebellar cortex (GCL-CCx): average area (S), numeric density (Nvs), and surface density (Sv) of synapses, and average volume (V), numeric density (Nvm), and volume density (Vv) of synaptic mitochondria. Moreover, succinic dehydrogenase (SDH) activity was cytochemically determined in Purkinje cells (PC) and V, Nvm, Vv, and cytochemical precipitate area/mitochondrial area (R) of SDH-positive mitochondria were evaluated. In SM CA1, MCT-KDs induced the early appearance of the morphological patterns typical of old animals: higher S and V, and lower Nvs and Nvm. On the contrary, in OML DG, Sv and Vv of MCT-KDs-fed rats were higher (as a result of higher Nvs and Nvm) vs. controls; these modifications are known to improve synaptic function and metabolic supply. The opposite effects of MCT-KDs might reflect the different susceptibility of these brain regions to the aging processes: OML DG is less vulnerable than SM CA1, and the reactivation of ketone bodies uptake and catabolism might occur more efficiently in this region, allowing the exploitation of their peculiar metabolic properties. In GCL-CCx, the results described a new scenario in comparison to that found in the hippocampal formation: 10%MCT-KD induced the early appearance of senescent patterns (decreased Nvs and Nvm; increased V), whereas 20%MCT-KD caused no changes. Since GCL-CCx is more vulnerable to age than DG, and less than CA1, these data further support the hypothesis that MCT-KDs effects in the aging brain critically depend on neuronal vulnerability to age, besides MCT percentage. Regarding PC, it was decided to evaluate only the metabolic effect of the dietetic regimen (20%MCT-KD) characterized by less side effects. KD counteracted age-related decrease in numeric density of SDH-positive mitochondria, and enhanced their energetic efficiency (R was significantly higher in MCT-KD-fed rats vs. all the controls). Since it is well known that Purkinje and dentate gyrus cells are less vulnerable to aging than CA1 neurons, these results corroborate our previous hypothesis. In conclusion, the A) experimental line provides the first evidence that morphological and functional parameters reflecting synaptic plasticity and mitochondrial metabolic competence may be modulated by MCT-KDs in the pre-senescent central nervous system, and that the effects may be heterogeneous in different brain regions. MCT-KDs seem to supply high energy metabolic intermediates and to be beneficial (“anti-aging”) for those neurons that maintain the capability to exploit them. This implies risks but also promising potentialities for the therapeutic use of these diets during aging B) Morphological parameters of synapses and synaptic mitochondria in SM CA1 were investigated in old (26-27 month-old) female Wistar rats following a single trial inhibitory avoidance task. In this memory protocol animals learn to avoid a dark compartment in which they received a mild, inescapable foot-shock. Rats were tested 3 and 6 or 9 hours after the training, divided into good and bad responders according to their performance (retention times above or below 100 s, respectively) and immediately sacrificed. Nvs, S, Sv, Nvm, V, and Vv were evaluated. In the good responder group, the numeric density of synapses and mitochondria was significantly higher and the average mitochondrial volume was significantly smaller 9 hours vs. 6 hours after the training. No significant differences were observed among bad responders. Thus, better performances in passive avoidance memory task are correlated with more efficient plastic remodeling of synaptic contacts and mitochondria in hippocampal CA1. These findings indicate that maintenance of synaptic plastic reactivity during aging is a critical requirement for preserving long-term memory consolidation

Peripheral Blood Brain-Derived Neurotrophic Factor as a Biomarker of Alzheimerâ\u80\u99s Disease: Are There Methodological Biases?

Mounting evidence that alterations in brain-derived neurotrophic factor (BDNF) levels and signaling may be involved in the etiopathogenesis of Alzheimer’s disease (AD) has suggested that its blood levels could be used as a biomarker of the disease. However, higher, lower, or unchanged circulating BDNF levels have all been described in AD patients compared to healthy controls. Although the reasons for such different findings are unclear, methodological issues are likely to be involved. The heterogeneity of participant recruitment criteria and the lack of control of variables that influence circulating BDNF levels regardless of dementia (depressive symptoms, medications, lifestyle, lack of overlap between serum and plasma, and experimental aspects) are likely to bias result and prevent study comparability. The present work reviews a broad panel of factors, whose close control could help reduce the inconsistency of study findings, and offers practical advice on their management. Research directed at elucidating the weight of each of these variables and at standardizing analytical methodologies is urgently needed

Effect of a Cognitive Training Program on the Platelet APP Ratio in Patients with Alzheimer’s Disease

In patients with Alzheimer’s disease (AD), synaptic plasticity seems to be involved in cognitive improvement induced by cognitive training. The platelet amyloid precursor protein (APP) ratio (APPr), i.e., the ratio between two APP isoforms, may be a useful peripheral biomarker to investigate synaptic plasticity pathways. This study evaluates the changes in neuropsychological/cognitive performance and APPr induced by cognitive training in AD patients participating in the “My Mind Project”. Neuropsychological/cognitive variables and APPr were evaluated in the trained group (n = 28) before a two-month experimental protocol, immediately after its termination at follow-up 1 (FU1), after 6 months at follow-up 2 (FU2), and after 24 months at follow-up 3 (FU3). The control group (n = 31) received general psychoeducational training for two months. Some memory and attention parameters were significantly improved in trained vs. control patients at FU1 and FU2 compared to baseline (Δ values). At FU3, APPr and Mini Mental State Examination (MMSE) scores decreased in trained patients. Δ APPr correlated significantly with the Δ scores of (i) MMSE at FU1, (ii) the prose memory test at FU2, and (iii) Instrumental Activities of Daily Living (IADL), the semantic word fluency test, Clinical Dementia Rating (CDR), and the attentive matrices test at FU3. Our data demonstrate that the platelet APPr correlates with key clinical variables, thereby proving that it may be a reliable biomarker of brain function in AD patients

Long-term visual object recognition memory in aged rats

Aging is associated with memory impairments, but the neural bases of this process need to be clarified. To this end, behavioral protocols for memory testing may be applied to aged animals to compare memory performances with functional and structural characteristics of specific brain regions. Visual object recognition memory can be investigated in the rat using a behavioral task based on its spontaneous preference for exploring novel rather than familiar objects. We found that a behavioral task able to elicit long-term visual object recognition memory in adult Long-Evans rats failed in aged (25–27 months old) Wistar rats. Since no tasks effective in aged rats are reported in the literature, we changed the experimental conditions to improve consolidation processes to verify that this form of memory can still be maintained for long term at this age: the learning trials were performed in a smaller box, identical to the home cage, and the inter-trial delays were shortened. We observed a reduction in anxiety in this box (as indicated by the lower number of fecal boli produced during habituation), and we found a learning protocol able to elicit a visual object recognition memory that was maintained after 24 h in these aged rats. When we applied the same protocol to adult rats, we obtained similar results. This experimental approach can be useful to study functional and structural changes associated with age-related memory impairments, and may help to identify new behavioral strategies and molecular targets that can be addressed to ameliorate memory performances during aging

Two Behavioral Tests Allow a Better Correlation Between Cognitive Function and Expression of Synaptic Proteins

The molecular substrate of age-associated cognitive decline (AACD) is still elusive. Evidence indicates that AACD is related to synaptic impairment in hippocampus, but different hippocampal regions play different roles, with the dorsal hippocampus (DH) associated to spatial learning, and the ventral hippocampus (VH) crucial for emotionality. If changes in hippocampal function contributes to AACD, this contribution may be reflected in alterations of synaptic protein levels. A commonly used approach to investigate this issue is western blotting. When this technique is applied to the entire hippocampus and the cognitive impairment is evaluated by a single task, changes in expression of a protein might undergo a “dilution effect”, as they may occur only in a given hippocampal region. We show that two behavioral tests yield more accurate results than one test in evaluating the function of the whole rat hippocampus by studying the expression of synaptotagmin 1 (SYT1), a vesicular protein whose expression in aged hippocampus is reportedly inconsistent. Analysis of SYT1 levels in the whole hippocampus of rats selected by the Morris water maze (MWM) test only failed to highlight a difference, whereas analysis of SYT1 levels in the whole hippocampus of rats categorized by both the MWM and the step-through passive avoidance (STPA) tests demonstrated a significant increase of SYT1 level in impaired rats. These findings, besides showing that SYT1 increases in impaired aged rats, suggest that using the whole hippocampus in blotting studies may prevent false negative results only if animals are categorized with tests exploring both DH and VH

Contribution of non-reference alleles in mtDNA of Alzheimer’s disease patients.

Many observations suggest that mutations of mitochondrial DNA (mtDNA) could be responsible for the neurodegenerative changes of Alzheimer's disease (AD). Here we examined the signal intensity of the four alleles of each mtDNA nucleotide position (np) in whole blood of AD patients and age-matched controls using MitoChip v2.0 array. Our analysis identified 270 significantly different nps which, with one exception, showed an increased contribution of non-reference alleles in AD patients. Principal component analysis (PCA) and cluster analysis showed that five of these nps could discriminate AD from control subjects with 80% of cases correctly classified. Our data support the hypothesis of mtDNA alterations as an important factor in the etiology of AD

MRI characterization of rat brain aging at structural and functional level: Clues for translational applications

Magnetic resonance imaging (MRI) paradigms, using non-invasive approaches, can provide relevant findings about brain aging. The attention has been primarily focused on neurodegenerative diseases, while little or nothing has been done to differentiate physiology from pathology. The present study aimed to test diffusion tensor imaging (DTI) and functional MRI (fMRI) metrics to analyze physiological age-related changes in rats at myelin structure and activation level; findings were validated by ex vivo histology. The purpose is to find comparable biomarkers in rodents and humans to allow a reliable translation from pre-clinical to clinical settings. Data evidenced: i) a significantly higher cerebrospinal fluid volume in middle-aged and aged vs. young rats; ii) a progressive alteration of white matter; iii) a significant reduction of evoked activity in aged animals. These results partially mirror the age-related changes in humans and may represent a preliminary step to find reliable tools for a lifelong monitoring with a value for the clinical practice (e.g., to provide support to the early diagnosis of dementia in asymptomatic subjects)

Bacterial DNAemia in Alzheimer’s Disease and Mild Cognitive Impairment: Association with Cognitive Decline, Plasma BDNF Levels, and Inflammatory Response

Microbial dysbiosis (MD) provokes gut barrier alterations and bacterial translocation in the bloodstream. The increased blood bacterial DNA (BB-DNA) may promote peripheral- and neuro-inflammation, contributing to cognitive impairment. MD also influences brain-derived neurotrophic factor (BDNF) production, whose alterations contribute to the etiopathogenesis of Alzheimer’s disease (AD). The purpose of this study is to measure BB-DNA in healthy elderly controls (EC), and in patients with mild cognitive impairment (MCI) and AD to explore the effect on plasma BDNF levels (pBDNF), the inflammatory response, and the association with cognitive decline during a two-year follow-up. Baseline BB-DNA and pBDNF were significantly higher in MCI and AD than in EC. BB-DNA was positively correlated with pBDNF in AD, plasma Tumor necrosis factor-alpha (TNF-α), and Interleukin-10 (IL-10) levels in MCI. AD patients with BB-DNA values above the 50th percentile had lower baseline Mini-Mental State Examination (MMSE). After a two-year follow-up, AD patients with the highest BB-DNA tertile had a worse cognitive decline, while higher BB-DNA levels were associated with higher TNF-α and lower IL-10 in MCI. Our study demonstrates that, in early AD, the higher the BB-DNA levels, the higher the pBDNF levels, suggesting a defensive attempt; BB-DNA seems to play a role in the AD severity/progression; in MCI, higher BB-DNA may trigger an increased inflammatory response