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Alzheimer's disease (AD) is the most prevalent type of dementia affecting older people. The identification of biomarkers is increasingly important and would be crucial for future therapy. Here, we demonstrated that in AD erythrocytes: (i) the anion transporter band3 is highly phosphorylated; (ii) the lyn kinase is phosphorylated and activated; (iii) the tyrosine phosphatase activity is downregulated, with a significant inverse correlation between band3 phosphorylation and disease progression, as revealed by Mini Mental State Examination score. Finally, we showed that in normal erythrocytes, treated in vitro with Aβ1-42 peptide, both band3 phosphorylation and lyn activation occurs. These results suggest that modulation of tyrosine phosphorylation signaling may be evaluated as a potential peripheral marker in AD.

Background Mild cognitive impairment (MCI) is usually described as an intermediate phase between normal cognition and dementia. Identifying the subjects at a higher risk of progressing from MCI to AD is essential to manage this condition. The diagnosis of MCI is mainly clinical. Several biomarkers have been proposed, but mostly for research purposes, as they are based on an invasive procedure to obtain the sample, such as cerebrospinal fluid (CSF). As a consequence, rapid and non-invasive biomarkers are needed to improve diagnosis. The objective of this systematic review is to summarize available evidence on the use of miRNAs as biomarkers in subjects with MCI. Methods Relevant literature published up to June 2018 was retrieved searching the databases PubMed, ISI Web of Knowledge and the Cochrane Database. Only studies considering microRNAs (miRNAs) and a diagnosis of MCI were included. Data were extracted using a specifically-designed standardized form, and their methodological quality was assessed using QUADAS-2 and QUIPS. Results Twenty-one studies of 153 retrieved articles met the predefined inclusion/exclusion criteria. Studies included participants ranging from 6 to 330. More than 40 miRNAs resulted as dysregulated, and miR-206 was the only miRNA that was found as differentially expressed in patients with MCI by more than two studies. However, these results have either not yet been confirmed in other independent cohorts, or data are still inconsistent. Inconsistencies among included studies could be due to several issues including the selection of participants, pre-analytical and analytical procedures, and statistical analyses.

Sex is a significant variable in the prevalence and incidence of neurological disorders. Sex differences exist in neurodegenerative disorders (NDs), where sex dimorphisms play important roles in the development and progression of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. In the last few years, some sex specific biomarkers for the identification of NDs have been described and recent studies have suggested that microRNA (miRNA) could be included among these, as influenced by the hormonal and genetic background. Failing to consider the possible differences between males and females in miRNA evaluation could introduce a sex bias in studies by not considering some of these sex-related biomarkers. In this review, we recapitulate what is known about the sex-specific differences in peripheral miRNA levels in neurodegenerative diseases. Several studies have reported sex-linked disparities, and from the literature analysis miR-206 particularly has been shown to have a sex-specific involvement. Hopefully, in the near future, patient stratification will provide important additional clues in diagnosis, prognosis, and tailoring of the best therapeutic approaches for each patient. Sex-specific biomarkers, such as miRNAs, could represent a useful tool for characterizing subgroups of patients.

Glioblastoma multiforme (GBM) is the most malignant brain tumor. Hypoxic condition is a predominant feature of the GBM contributing to tumor growth and resistance to conventional therapies. Hence, the identification of drugs able to impair GBM malignancy and aggressiveness is considered of great clinical relevance. Previously, we demonstrated that the activation of M2 muscarinic receptors, through the agonist arecaidine propargyl ester (Ape), arrests cell proliferation in GBM cancer stem cells (GSCs). In the present work, we have characterized the response of GSCs to hypoxic condition showing an upregulation of hypoxia-inducible factors and factors involved in the regulation of GSCs survival and proliferation. Ape treatment in hypoxic conditions is however able to inhibit cell cycle progression, causing a significant increase of aberrant mitosis with consequent decreased cell survival. Additionally, qRT-PCR analysis suggest that Ape downregulates the expression of stemness markers and miR-210 levels, one of the main regulators of the responses to hypoxic condition in different tumor types. Our data demonstrate that Ape impairs the GSCs proliferation and survival also in hypoxic condition, negatively modulating the adaptive response of GSCs to hypoxia.

Objective: To report, for the first time, a large autosomal dominant Alzheimer disease (AD) family in which the APP A713T mutation is present in the homozygous and heterozygous state. To date, the mutation has been reported as dominant, and in the heterozygous state associated with familial AD and cerebrovascular lesions. Methods: The family described here has been genealogically reconstructed over 6 generations dating back to the 19th century. Plasma β-amyloid peptide was measured. Sequencing of causative AD genes was performed. Results: Twenty-one individuals, all but 1 born from 2 consanguineous unions, were studied: 8 were described as affected through history, 5 were studied clinically and genetically, and 8 were asymptomatic at-risk subjects. The A713T mutation was detected in the homozygous state in 3 patients and in the heterozygous state in 8 subjects (6 asymptomatic and 2 affected). Conclusions: Our findings, also supported by the β-amyloid plasma assay, confirm (1) the pathogenic role of the APP A713T mutation, (2) the specific phenotype (AD with cerebrovascular lesions) associated with this mutation, and (3) the large span of age at onset, not influenced by APOE , TOMM40 , and TREM2 genes. No substantial differences concerning clinical phenotype were evidenced between heterozygous and homozygous patients, in line with the classic definition of dominance. Therefore, in this study, AD followed the classic definition of a dominant disease, contrary to that reported in a previously described AD family with recessive APP mutation. This confirms that genetic AD may be considered a disease with dominant and recessive traits of inheritance.

We have recently isolated a new isoform of recombinant manganese superoxide dismutase (rMnSOD) which provides a potent antitumor activity and strongly counteracts the occurrence of oxidative stress and tissue inflammation. This isoform, in addition to the enzymatic action common to all SODs, also shows special functional and structural properties, essentially due to the presence of a first leader peptide that allows the protein to enter easily into cells. Among endogenous antioxidants, SOD constitutes the first line of natural defence against pathological effects induced by an excess of free radicals. Here, we firstly describe the effects of our rMnSOD administration on the proliferant and malignant undifferentiated human neuroblastoma SK-N-BE cell line. Moreover, we also test the effects of rMnSOD in the all trans retinoic-differentiated SK-N-BE neuron-like cells, a quiescent “not malignant” model. While rMnSOD showed an antitumor activity on proliferating cells, a poor sensitivity to rMnSOD overload in retinoid-differentiated neuron-like cells was observed. However, in the latter case, in presence of experimental-induced oxidative stress, overcharge of rMnSOD enhanced the oxidant effects, through an increase of H2O2 due to low activity of both catalase and glutathione peroxidase. In conclusion, our data show that rMnSOD treatment exerts differential effects, which depend upon both cell differentiation and redox balance, addressing attention to the potential use of the recombinant enzyme on differentiated neurons. These facts ultimately pave the way for further preclinical studies aimed at evaluation of rMnSOD effects in models of neurodegenerative diseases.

Given the heterogeneous nature of frontotemporal dementia (FTD), sensitive biomarkers are greatly needed for the accurate diagnosis of this neurodegenerative disorder. Circulating miRNAs have been reported as promising biomarkers for neurodegenerative disorders and processes affecting the central nervous system, especially in aging. The objective of the study was to evaluate if some circulating miRNAs linked with apoptosis (miR-29b-3p, miR-34a-5p, miR-16-5p, miR-17-5p, miR-107, miR-19b-3p, let-7b-5p, miR-26b-5p, and 127-3p) were able to distinguish between FTD patients and healthy controls. For this study, we enrolled 127 subjects, including 54 patients with FTD, 20 patients with Alzheimer's disease (AD), and 53 healthy controls. The qRT-PCR analysis showed a downregulation of miR-127-3p in FTD compared to controls, while the levels of other miRNAs remained unchanged. Then, miR-127-3p expression was also analyzed in AD patients, finding a different expression between two patient groups. A receiver operating characteristic curve was then created for miR-127-3p to discriminate FTD versus AD (AUC: 0.8986), and versus healthy controls (AUC: 0.8057). In conclusion, miR-127-3p could help to diagnose FTD and to distinguish it from AD.

An expanded hexanucleotide (GGGGCC) repeat in a non-coding promoter region of open reading frame 72 of chromosome 9 (C9ORF72) has been recently identified as a major cause of familial and sporadic frontotemporal lobar degeneration. We describe the clinical picture of a 64-year-old woman carrying the hexanucleotide repeat expansion, who developed a sporadic early-onset form of behavioral variant frontotemporal dementia characterized by the occurrence of uncommon behavioral manifestations such as binge eating disturbance and by a rapid worsening of cognitive abilities. Our report confirms previous studies asserting that C9ORF72 repeats may sustain heterogeneous clinical syndromes.

The purpose of this study was to develop an easy and minimally invasive assay to detect a plasma miRNA profile in frontotemporal dementia (FTD) patients, with the final aim of discriminating between FTD patients and healthy controls (HCs). After a global miRNA profiling, significant downregulation of miR-663a, miR-502-3p, and miR-206 (p = 0.0001, p = 0.0002, and p = 0.02 respectively) in FTD patients was confirmed when compared with HCs in a larger case-control sample. Moreover, miR-663a and miR-502-3p showed significant differences in both genders, whereas miR-206, only in male subjects. To obtain a discriminating measure between FTD patients and HCs, we calculated a combined score of the 3 miRNAs by applying a Bayesian approach and obtaining a classifier with an accuracy of 84.4%. Moreover, for men, combined miRNA levels showed an excellent sensitivity (100%) and a good specificity (87.5%) in distinguishing FTD patients from HCs. All these findings open new hypotheses in the pathophysiology and new perspectives in the diagnosis of a complex pathology as FTD.

Among promising biological markers proposed for Parkinson's disease (PD) and other disorders related to Lewy bodies, plasma alpha-synuclein assay has provided conflicting results mainly owing to the various laboratory assay techniques used and protein forms assayed. In this observational and exploratory cross-sectional study, using an immunoenzymatic technique, we assayed and compared total plasma alpha-synuclein concentrations in 69 patients with PD and 110 age-matched healthy control subjects. Two previously unreported findings concerned gender. First, plasma alpha-synuclein concentrations measured in the more advanced parkinsonian disease stages decreased in men, but not in women. Second, again only in men, plasma alpha-synuclein concentration was associated with cognitive impairments, hallucinations, and sleep disorders. These findings underline the gender-related differences in parkinsonian patients and indicate plasma alpha-synuclein expression as a potential biological marker for PD progression in men.

Despite considerable advances in the treatment of human inflammatory diseases, such as cardiovascular and neurological disorders, they remain the leading cause of death in developed countries. From a clinical perspective, an active area of investigation focuses on the identification of diagnostic and prognostic biomarkers, because preventing events in those at risk of chronic inflammatory disease is likely to have a substantial impact on the overall public-health burden. The sex difference has not been considered previously as important in the evaluation of biomarkers of human diseases, notwithstanding it is now ascertained that the severity of these disorders is correlated with sex hormones which modulate the inflammatory response. The aim of the present brief review is to report and comment the state of art regarding the sex-related biomarkers in cardiovascular and neurodegenerative disorders, focusing on those compounds showing potential prognostic- and diagnostic values, and/or acting as indicators of the therapeutic treatment efficacy.

Progranulin (PGRN) is a secreted protein expressed ubiquitously throughout the body, including the brain, where it localizes in neurons and is activated microglia. Loss-of-function mutations in the GRN gene are an important cause of familial frontotemporal lobar degeneration (FTLD). PGRN has a neurotrophic and anti-inflammatory activity, and it is neuroprotective in several injury conditions, such as oxygen or glucose deprivation, oxidative injury, and hypoxic stress. Indeed, we have previously demonstrated that hypoxia induces the up-regulation of GRN transcripts. Several studies have shown microRNAs (miRNAs) involvement in hypoxia. Moreover, in FTLD patients with a genetic variant of GRN (rs5848), the reinforcement of miR-659-3p binding site has been suggested to be a risk factor. Here, we report that miR-659-3p interacts directly with GRN 3'UTR as shown by luciferase assay in HeLa cells and ELISA and Western Blot analysis in HeLa and Kelly cells. Moreover, we demonstrate the physical binding between GRN mRNA and miR-659-3p employing a miRNA capture-affinity technology in SK-N-BE and Kelly cells. In order to study miRNAs involvement in hypoxia-mediated up-regulation of GRN, we evaluated miR-659-3p levels in SK-N-BE cells after 24 h of hypoxic treatment, finding them inversely correlated to GRN transcripts. Furthermore, we analyzed an animal model of asphyxia, finding that GRN mRNA levels increased at post-natal day (pnd) 1 and pnd 4 in rat cortices subjected to asphyxia in comparison to control rats and miR-659-3p decreased at pnd 4 just when GRN reached the highest levels. Our results demonstrate the interaction between miR-659-3p and GRN transcript and the involvement of miR-659-3p in GRN up-regulation mediated by hypoxic/ischemic insults.

Frontotemporal lobar degeneration (FTLD) includes a spectrum of disorders characterized by changes of personality and social behavior and, often, a gradual and progressive language dysfunction. In the last years, several efforts have been fulfilled in identifying both genetic mutations and pathological proteins associated with FTLD. The molecular bases undergoing the onset and progression of the disease remain still unknown. Recent literature prompts an involvement of RNA metabolism in FTLD, particularly microRNAs (miRNAs). Dysregulation of miRNAs in several disorders, including neurodegenerative diseases, and increasing importance of circulating miRNAs in different pathologies has suggested to implement the study of their possible application as biological markers and new therapeutic targets; moreover, miRNA-based therapy is becoming a powerful tool to deepen the function of a gene, the mechanism of a disease, and validate therapeutic targets. Regarding FTLD, different studies showed that miRNAs are playing an important role. For example, several reports have evaluated miRNA regulation of the progranulin gene suggesting that it is under their control, as described for miR-29b, miR-107, and miR-659. More recently, it has been demonstrated that TMEM106B gene, which protein is elevated in FTLD-TDP brains, is repressed by miR-132/212 cluster; this post-transcriptional mechanism increases intracellular levels of progranulin, affecting its pathways. These findings if confirmed could suggest that these microRNAs have a role as potential targets for some related-FTLD genes. In this review, we focus on the emerging roles of the miRNAs in the pathogenesis of FTLD.

Objective: Screening plasma samples from patients with sporadic Creutzfeldt-Jakob disease (CJD) to discover diagnostic biomarkers. Methods: Plasma samples were collected from 17 patients with sporadic CJD, 17 patients with Alzheimer disease (AD), and 20 healthy subjects. A 2-phase screening was carried out using quantitative protein mass spectrometry. The putative sporadic CJD biomarkers were then validated independently by immunoturbidimetry. Results: Mass spectrometry uncovered 7 candidate sporadic CJD protein biomarkers, all belonging to the acute-phase response. Highly significant increases of these markers in patients with sporadic CJD, compared with healthy subjects and patients with AD, was confirmed by immunoturbidimetry. Conclusions: The increase in plasma levels of a related set of acute-phase reactants in patients with sporadic CJD is a novel finding that suggests new pathogenetic hypotheses. The possible value of this set of proteins as biomarkers in the diagnosis of sporadic CJD or for blood/tissue donor screening remains to be further explored and validated in larger studies.

Progranulin (PGRN) is a widely expressed multifunctional protein, involved in regulation of cell growth and cell cycle progression with a possible involvement in neurodegeneration. We looked for PGRN regulation in three different human neuroblastoma cell lines, following exposure to two different stimuli commonly associated to neurodegeneration: hypoxia and oxidative stress. For gene and protein expression analysis we carried out a quantitative RT-PCR and western blotting analysis. We show that PGRN is strongly up-regulated by hypoxia, through the mitogen-actived protein kinase (MAPK)/extracellular signal-regulated kinase (MEK) signaling cascade. PGRN is not up-regulated by H2O2-induced oxidative stress. These results suggest that PGRN in the brain could exert a protective role against hypoxic stress, one of principal risk factors involved in frontotemporal dementia pathogenesis.

The missense P39L variant in the prion protein gene (PRNP) has recently been associated with frontotemporal dementia (FTD). Here, we analyzed the presence of the P39L variant in 761 patients with FTD and 719 controls and found a single carrier among patients. The patient was a 67-year-old male, with a positive family history for dementia, who developed apathy, short term memory deficit, and postural instability at 66. Clinical and instrumental workup excluded prion disease. At MRI, bilateral frontal lobe atrophy was present. A diagnosis of FTD was made, with a mainly apathetic phenotype. The PRNP P39L mutation may be an extremely rare cause of FTD (0.13%).

Presenilin-1 (PSEN-1) is a component of the gamma-secretase complex involved in beta-amyloid precursor protein (betaAPP) processing. To date about 140 pathogenic mutations in the PSEN-1 gene have been identified and their main biochemical effect is to increase the production of the fibrillogenic peptide Abeta(1-42). An exception is the PSEN-1 [E318G] mutation that does not alter Abeta(1-42) generation and is generally considered a non-pathogenic polymorphism. Nevertheless, this mutation was reported to be a genetic risk factor for familial Alzheimer's disease (FAD) in the Australian population. To independently confirm this indication, we performed a case-control association study in the Italian population. We found a significant association (p0.05, Fisher's exact test) between the presence of PSEN-1 [E318G] and FAD. In addition, on measuring the Abeta(1-42) and Abeta(1-40) concentrations in fibroblast-conditioned media cultured from PSEN-1 [E318G] carriers and PSEN-1 [wild type] controls we noted a significant decrease (p0.05, Mann-Whitney test) in the Abeta(1-42)/Abeta(1-40) ratio in PSEN-1 [E318G] carriers, suggesting a peculiar biochemical effect of this mutation.

Background: Alzheimer’s disease (AD) is the most frequent progressive neurodegenerative disease. Cholinergic dysfunction is one of the major pathological alteration, although depletion of cholinergic neurons is caused by the well-established toxicity of the beta-amyloid plaques and neurofibrillary tangles. Cholinergic dysfunctions are consequences of the decrease in acetylcholine synthesis and release, and altered function of muscarinic and nicotinic cholinergic receptors. In addition, a direct correlation between cholinergic alteration, amyloidbeta production and tau phosphorylation, two main AD-pathology hallmarks, has been identified. Methods: In the present review we focused our discussion on the identification of new allosteric or bitopic ligands able to modulate the cholinergic receptor activity. Moreover drug delivery methodology (nanoparticeles, liposomes, etc.) that might contribute to drive the drug in the brain, reducing their toxicity and potential side effects have been also discussed. Results: Many drugs are currently in use for AD (e.g. donepezil, rivastigmine etc.) and several of those in development such as muscarininc and nicotinic agonists, target specifically the cholinergic system; the main mechanism aims to rescue the cholinergic dysfunction, to reduce neurotoxic protein accumulation and improve the cholinergic impairments responsible of the cognitive deficits. Promising approaches aim to either improve drug delivery into the brain or develope new compounds targeting known or new molecular pathways. Nanoparticles and liposomes are also described as new nanotechnology tools that overcome traditional routes of administration, with a particular focus on their employment for compounddelivery that targets the cholinergic system. Ultimately, a new fields of research is emerging as the use of induced pluripotent stem cells, a technology that allows to obtain cells directly from the patients that can be propagated indefinetely and differentiated into the susceptible neuronal subtypes. This may significantly contribute to improve the understanding of AD pathological processes and enhance current AD pharmacology beyond the cholinergic dysfunction. Conclusion: From the topics discussed in the present review, emerges that the combination between pharmacological studies and nanotechnological approaches for drug delivery and the identification of new specific models may largely enhance and improve the therapeutic strategies for different neurological disease including AD.

Neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), are caused by a combination of events that impair normal neuronal function. Although they are considered different disorders, there are overlapping features among them from the clinical, pathological, and genetic points of view. Synaptic dysfunction and loss, neurite retraction, and the appearance of other abnormalities such as axonal transport defects normally precede the neuronal loss that is a relatively late event. The diagnosis of many neurodegenerative diseases is mainly based on patient's cognitive function analysis, and the development of diagnostic methods is complicated by the brain's capacity to compensate for neuronal loss over a long period of time. This results in the late clinical manifestation of symptoms, a time when successful treatment is no longer feasible. Thus, a noninvasive diagnostic method based on early events detection is particularly important. In the last years, some biomarkers expressed in human body fluids have been proposed. microRNAs (miRNAs), with their high stability, tissue- or cell type-specific expression, lower cost, and shorter time in the assay development, could constitute a good tool to obtain an early disease diagnosis for a wide number of human pathologies, including neurodegenerative diseases. The possibilities and challenges of using these small RNA molecules as a signature for neurodegenerative disorders is a highly promising approach for developing minimally invasive screening tests and to identify new therapeutic targets.

We describe the genetic analysis of an Alzheimer's disease (AD) sample derived from a genetically isolated population. Genetic assessment included the analysis of genes involved in AD, such as the genes for amyloid precursor protein (APP), presenilin 1 (PSEN1) and presenilin 2 (PSEN2). We also assessed genes for some proteins that constitute the γ-secretase complex: nicastrin (NCSTN), presenilin enhancer-2 (PEN2), in addition to the AD risk factor apolipoprotein E (APOE). Using polymerase chain reaction and single strand conformational polymorphism method, screens for APP, PSEN1 and PSEN2 genes revealed one mutation in PSEN1. Furthermore, we found an intronic +17G > C polymorphism in PEN2 which, in homozygous form, was greater in early onset Alzheimer's disease (EOAD) compared to controls, and one haplotype in the NCSTN gene which was linked to EOAD and familial AD (FAD). Finally, the genotyping of APOE confirmed that the ɛ4 allele could be a risk factor for the onset of AD, in particular for FAD subjects. In conclusion, these results show the existence of Sardinian genetic peculiarities, essential in studies regarding genetically inherited and multifactorial disorders, as AD.

The neuropsychological assessment of non-demented subjects with gene mutation of familial Alzheimer’s disease (AD) provides a model for exploring the early cognitive features of the disease. We evaluated 1 patient and 6 non-demented subjects belonging to a family with AD with M239V mutation of the presenilin 2 gene, aiming to verify the contribution of specific cognitive patterns to the characterization of familial AD. One patient, 3 non-demented subjects with M239V mutation and 3 subjects without mutation from the same family underwent neuropsychological testing. The patient’s cognitive profile was characterized by anosognosia, visuospatial agnosia, apraxia and fluent aphasia. Of the 3 non-demented subjects with mutation, 1 showed no deficits, another constructive apraxia and the third spatial perception and memory deficits. The 3 subjects without mutation showed normal abilities. The cognitive deficits of the non-demented subjects with mutations indicate focal dysfunction of the posterior cortical areas, resembling the more extended parieto-occipito-temporal dysfunction of the demented patient. Such grading of visuospatial, praxis, and language impairments highlights a distinctive pattern related to the M239V mutation of the presenilin 2 gene.

Nicastrin is a type 1 transmembrane glycoprotein that interacts with presenilin, Aph-1, and Pen-2 proteins to form a high molecular complex with gamma secretase activity. Then, nicastrin has a central role in presenilin-mediated processing of beta-amyloid precursor protein and in some aspects of Notch/glp-1 signaling in vivo. Here, we isolated a rat nicastrin cDNA and investigated gene expression in embryonic and adult rat tissues. The predicted amino acid sequence is comprised of 708 residues and showed a high degree of identity with other vertebrate orthologs. Besides full-length nicastrin mRNA, we identified an alternative spliced variant lacking the whole exon 3 and predicted to encode a 62-residue-long truncated protein. Full-length nicastrin mRNA was observed to be ubiquitously expressed, while the spliced variant was preferentially transcribed in the nervous system, whether in embryonic or adult neural tissues. Studies performed on primary cell cultures demonstrated that the short isoform was expressed in neurons, but not in astrocyte and microglial cells. Further experiments performed to verify the presence of the variant in neuroblastoma culture failed to show any truncated protein. Treatments by cyclohexamide showed the involvement of a quality control-based surveillance mechanism, which selectively degrades the exon 3-skipped isoform. In summary, this is the first report describing a novel skipped isoform of nicastrin which may suggest a new possible control mechanism based on the alternative splicing and nonsense-mediated mRNA decay to regulate brain protein expression and provide newer insights into potential implication in Alzheimer's disease.

Genetic evidence indicates a central role of cerebral accumulation of beta-amyloid (Abeta) in the pathogenesis of Alzheimer's disease (AD). Beside presenilin 1 and 2, three other recently discovered proteins (Aph 1, PEN 2 and nicastrin) are associated with gamma-secretase activity, the enzymatic complex generating Abeta. Alterations in genes encoding these proteins were candidates for a role in AD. The PEN 2 gene was examined for unknown mutations and polymorphisms in sporadic and familial Alzheimer patients. Samples from age-matched controls (n=253), sporadic AD (SAD, n=256) and familial AD (FAD, n=140) were screened with DHPLC methodology followed by sequencing. Scanning the gene identified for the first time a missense mutation (D90N) in a patient with FAD. Three intronic polymorphisms were also identified, one of which had a higher presence of the mutated allele in AD subjects carrying the allele epsilon4 of apolipoprotein E than controls. The pathogenic role of the PEN-2 D90N mutation in AD is not clear, but the findings might lead to new studies on its functional and genetic role.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease whose etiology is still obscure. The most common mutations found in familiar ALS are localized in the gene codifying the copper-zinc superoxide dismutase 1 (SOD1) enzyme which, however, explains only about 20% of familiar ALS and 2% of the sporadic form of this disease.

Neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), are caused by a combination of events that impair normal neuronal function. Although they are considered different disorders, there are overlapping features among them from the clinical, pathological, and genetic points of view. Synaptic dysfunction and loss, neurite retraction, and the appearance of other abnormalities such as axonal transport defects normally precede the neuronal loss that is a relatively late event. The diagnosis of many neurodegenerative diseases is mainly based on patient’s cognitive function analysis, and the development of diagnostic methods is complicated by the brain’s capacity to compensate for neuronal loss over a long period of time. This results in the late clinical manifestation of symptoms, a time when successful treatment is no longer feasible. Thus, a noninvasive diagnostic method based on early events detection is particularly important. In the last years, some biomarkers expressed in human body fluids have been proposed. microRNAs (miRNAs), with their high stability, tissue- or cell type-specific expression, lower cost, and shorter time in the assay development, could constitute a good tool to obtain an early disease diagnosis for a wide number of human pathologies, including neurodegenerative diseases. The possibilities and challenges of using these small RNA molecules as a signature for neurodegenerative disorders is a highly promising approach for developing minimally invasive screening tests and to identify new therapeutic targets.

We have shown previously that the amyloid precursor protein (APP) is synthesized in retinal ganglion cells and is rapidly transported down the axons, and that different molecular weight forms of the precursor have different developmental time courses. Some APP isoforms contain a Kunitz protease inhibitor (KPI) domain, and APP that lacks the KPI domain is considered the predominant isoform in neurons. We now show that, among the various rapidly transported APPs, a 140-kDa isoform contains the KPI domain. This APP isoform is highly expressed in rapidly growing retinal axons, and it is also prominent in adult axon endings. This 140-kDa KPI-containing APP is highly sulfated compared with other axonally transported isoforms. These results show that APP with the KPI domain is a prominent isoform synthesized in neurons in vivo, and they suggest that the regulation of protease activity may be an important factor during the establishment of neuronal connections.

Neurodegenerative disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and frontotemporal dementias (FTD), are considered distinct entities, however, there is increasing evidence of an overlap from the clinical, pathological and genetic points of view. All neurodegenerative diseases are characterized by neuronal loss and death in specific areas of the brain, for example, hippocampus and cortex for AD, midbrain for PD, frontal and temporal lobes for FTD. Loss of neurons is a relatively late event in the progression of neurodegenerative diseases that is typically preceded by other events such as metabolic changes, synaptic dysfunction and loss, neurite retraction, and the appearance of other abnormalities, such as axonal transport defects. The brain’s ability to compensate for these dysfunctions occurs over a long period of time and results in late clinical manifestation of symptoms, when successful pharmacological intervention is no longer feasible. Currently, diagnosis of AD, PD and different forms of dementia is based primarily on analysis of the patient’s cognitive function. It is therefore important to find non-invasive diagnostic methods useful to detect neurodegenerative diseases during early, preferably asymptomatic stages, when a pharmacological intervention is still possible. Altered expression of microRNAs (miRNAs) in many disease states, including neurodegeneration, and increasing relevance of miRNAs in biofluids in different pathologies has prompted the study of their possible application as neurodegenerative diseases biomarkers in order to identify new therapeutic targets. Here, we review what is known about the role of miRNAs in the pathogenesis of neurodegeneration and the possibilities and challenges of using these small RNA molecules as a signature for neurodegenerative conditions.

Previous immunohistochemical studies in mouse, rat, and chick have reported that the expression of the glycoprotein and cell adhesion molecule L1, a member of the immunoglobulin superfamily, shows regulation during development of retina and optic nerve. To extend our understanding of the role of L1 in developing neural circuitry, we have examined L1 expression in the optic tract and thalamic and midbrain synaptic targets of retinal fibers in the early postnatal Syrian hamster, a well-characterized developmental model of the primary visual projection. Metabolic labeling studies reveal that a synaptically targeted, sulfated, and glycosylated form of L1 undergoes rapid axonal transport from the retina. Retinofugal transport of L1 decreases commensurate with the decline in immunoreactivity of retinal fibers in the visual pathway. Retinal ganglion cell axons show intense L1 immunoreactivity as they navigate in highly fasciculated bundles in the optic tract overlying the lateral geniculate body and in the superior colliculus. We found no evidence of L1 immunoreactivity on retinal axon collaterals as they defasciculate from the optic tract and branch into target neuropils. L1 immunoreactivity wanes in optic tract as axon terminal arbors are elaborated in the lateral geniculate body and superior colliculus and as myelination in the visual pathway commences. This pattern of L1 expression suggests that, in the early postnatal period, L1 may support fasciculation of retinal fibers, maintaining them within the optic tract, and that subsequent down-regulation of L1 may facilitate their terminal arborization and myelination.

The amyloid precursor superfamily is composed of three highly conserved transmembrane glycoproteins, the amyloid precursor protein (APP) and amyloid precursor-like proteins 1 and 2 (APLP1, APLP2), whose functions are unknown. Proteolytic cleavage of APP yields the betaA4 peptide, the major component of cerebral amyloid in Alzheimer's disease. Here we show that five post-translationally modified, full-length species of APP and APLP2 (but not APLP1) arrive at the mature presynaptic terminal in the fastest wave of axonal transport and are subsequently rapidly cleared (mean half-life of 3.5 h). Rapid turnover of presynaptic APP and APLP2 occurs independently of visual activity. Turnover of the most rapidly arriving APP species was accompanied by a delayed accumulation of a 120-kDa, APP fragment lacking the C terminus, consistent with presynaptic APP turnover via constitutive proteolysis. Turnover of APLP2 was not accompanied by detectable APLP2 fragment peptides, suggesting either that APLP2 either is more rapidly degraded than is APP or is retrogradely transported shortly after reaching the terminus. A single 150-kDa APLP2 species containing the Kunitz protease inhibitor domain is the major amyloid precursor superfamily protein transported to the presynapse. Presynaptic APP and APLP2 are sialylated and N- and O-glycosylated, and some also carry chondroitin sulfate glycosaminoglycan and/or dermatan sulfate glycosaminoglycan. The rapid kinetics for turnover of APP and APLP2 predict a sensitive balance of synthesis, transport, and elimination rates that may be critical to normal neuronal functions and metabolic fates of these proteins.

Synaptic vesicle protein 2 is an integral synaptic vesicle membrane glycoprotein which is present in all synapses for which it has been examined. We used an anti-synaptic vesicle protein 2 monoclonal antibody to examine synaptic vesicle protein 2 localization in the developing hamster retinofugal pathway. From postnatal day 0 to day 1, a period of elongation of retinal ganglion cell axons to their central targets, fiber fascicles in the optic tract over the lateral geniculate nucleus were intensely synaptic vesicle protein 2-immunoreactive. Adjacent to the optic tract, single fibers could be seen. We also observed a marked immunostaining in growth cones and fiber fascicles in retinal explants in culture. By postnatal day 2, the staining of single fibers had ended, and by postnatal day 5, during the formation of terminal arbors, numerous fine puncta of synaptic vesicle protein 2 immunoreactivity were distributed within the neuropil of the lateral geniculate nucleus. In the adult, the optic tract was devoid of synaptic vesicle protein 2 staining, while the neuropil contained distinct immunoreactive profiles, particularly in the outer shell of the lateral geniculate. These synaptic vesicle protein 2-positive profiles closely resembled the grape-like clusters and large swellings of two known retinal axon terminal types. Eye removal resulted in the rapid disappearance of these synaptic vesicle protein 2-labelled terminal profiles contralateral to the enucleation. A similar pattern of synaptic vesicle protein 2 immunoreactivity was observed in the superior colliculus. From postnatal day 0 to day 2, retinal fiber fascicles in the stratum griseum superficiale/stratum opticum were darkly stained for synaptic vesicle protein 2. By postnatal day 5, the immunoreactivity shifted to the neuropil and from postnatal day 6 onwards, the synaptic vesicle protein 2 immunoreactivity was more intense in the stratum griseum superficiale than in the optic fibre layer. This study demonstrates dense synaptic vesicle protein 2-labelling of elongating axons both in vivo and in vitro. However, coincident with the transition from retinal ganglion cell axon elongation to terminal arborization, synaptic vesicle protein 2 is progressively restricted to synaptic terminals and becomes undetectable in axons. This study is the first to document an axonal localization of synaptic vesicle protein 2 during development and raises the question as to its role during axonal elongation.

Familial Alzheimer's disease (FAD), despite representing a rare condition, is attracting a growing interest in the scientific community. Improved phenotyping of FAD cases may have a relevant impact both in clinical and research contexts. We performed a systematic review of studies describing the phenotypic features of FAD cases sustained by PSEN2 mutations, the less common cause of monogenic AD. Special attention was given to the clinical manifestations as well as to the main findings coming from the most commonly and widely adopted diagnostic procedures. Basing on the collected data, we also attempted to conduct a genotype-phenotype correlation analysis. Overall, the mutations involving the PSEN2 gene represent an extremely rare cause of FAD, having been reported to date in less than 200 cases. They are mainly associated, despite some peculiar and heterogeneous features, to a typical AD phenotype. Nevertheless, the frequent occurrence of psychotic symptoms may represent a potential distinctive element. The scarcity of available phenotypic descriptions strongly limits the implementation of genotype-phenotype correlations.

Presenilin 1 and presenilin 2 are widely expressed during brain development. Several mutations in these proteins have been associated with autosomal-dominant inherited forms of Alzheimer disease. Their expression is regulated by various cellular and extracellular factors, which change with age and sex. Both age and sex are key risk factors for Alzheimer’s disease, but the issue of whether the expression of presenilins is influenced by the sex during early postnatal development of the brain has been poorly investigated so far. In this study, we report that transcript levels of presenilins, and the subset of neurons expressing these proteins in various brain areas of the developing post-natal brain are different in male and female rats, suggesting that their function(s) may contribute to sexual dimorphism in the brain, both at morphological and functional levels.

Background: A hexanucleotide repeat expansion in the first intron of C9ORF72 has been shown to be responsible for a high number of familial cases of amyotrophic lateral sclerosis or frontotemporal lobar degeneration (FTLD). Atypical presentations have been described, particularly psychosis. Methods: We determined the frequency of the hexanucleotide repeat expansions in a population of 651 FTLD patients and compared the clinical characteristics of carriers and noncarriers. In addition, we genotyped 21 patients with corticobasal syndrome, 31 patients with progressive supranuclear palsy, and 222 control subjects. Results: The pathogenic repeat expansion was detected in 39 (6%) patients with FTLD (17 male and 22 female subjects); however, it was not detected in any corticobasal syndrome and progressive supranuclear palsy patients or controls. Twenty-four of 39 carriers had positive family history for dementia and/or amyotrophic lateral sclerosis (61.5%), whereas only 145 of 612 noncarriers had positive family history (23.7%; p

Plasma progranulin (PGRN) levels constitute a potentially invaluable biomarker for neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and, perhaps, Alzheimer's disease (AD). We assessed plasma PGRN levels in 107 AD patients, 36 FTLD patients, and 107 controls. We found that, in female AD patients, there is a positive correlation between PGRN levels and age. Although no significant differences were found between patients and controls, we observed higher levels in females compared to males; in AD patients, a positive correlation between PGRN levels and age was observed in females. In conclusion, our data suggest that PGRN may not be a good biomarker for AD; moreover, gender may influence the plasma PGRN levels of AD patients.

Presenilin-1 (PS1) and presenilin-2 (PS2) are transmembrane proteins widely expressed in the central nervous system, which function as the catalytic subunits of γ-secretase, the enzyme that releases amyloid-β protein (Aβ) from ectodomain cleaved amyloid precursor protein (APP) by intramembrane proteolysis. Mutations in PS1, PS2, and Aβ protein precursor are involved in the etiology of familial Alzheimer’s disease (FAD), while the cause of the sporadic form of AD (SAD) is still not known. However, since similar neuropathological changes have been observed in both FAD and SAD, a common pathway in the etiology of the disease has been suggested. Given that age-related deranged Ca2+ regulation has been hypothesized to play a role in SAD pathogenesis via PS gene regulation and γ-secretase activity, we studied the in vitro regulation of PS1 and PS2 in the human neuron-like SK-N-BE cell line treated with the specific endoplasmic reticulum (ER) calcium ATPase inhibitor Thapsigargin (THG), to introduce intracellular Ca2+ perturbations and mimic the altered Ca2+ homeostasis observed in AD. Our results showed a consistent and significant down-regulation of PS2, while PS1 appeared to be unmodulated. These events were accompanied by oxidative stress and a number of morphological alterations suggestive of the induction of apoptotic machinery. The administration of the antioxidant N-acetylcysteine (NAC) did not revert the THG-induced effects reported, while treatment with the Ca2+-independent ER stressor Brefeldin A did not modulate basal PS1 and PS2 expression. Collectively, these results suggest that Ca2+ fluctuation rather than ER stress and/or oxidative imbalance seems to play an essential role in PS2 regulation and confirm that, despite their strong homology, PS1 and PS2 could play different roles in AD.

Alzheimer's disease (AD) is a neurodegenerative disorder often treated with donepezil, an acetylcholinesterase inhibitor. Response to donepezil is variable, probably based on patients' genetic background in donepezil metabolizing enzymes, including cytochrome 2D6 (CYP2D6). We evaluated the association between clinical response to donepezil and a common variant (rs1080985) of CYP2D6, previously reported to be associated with poor response to the drug. In a sample of 415 AD cases, we found evidence of association between rs1080985 and response to donepezil after 6 months of therapy (OR [95% CI]: 1.74 [1.01-3.00], p = 0.04). Rs1080985 might be useful as predictor of poor response to short-term donepezil treatment.

Autophagy, the cytoprotection mechanism that takes place under metabolic impairment, has been implicated in the pathogenesis of autoimmunity. Here, we investigated the spontaneous and induced autophagic behavior of T lymphocytes from patients with systemic lupus erythematosus (SLE) compared with that of T lymphocytes from healthy donors by measuring the autophagy marker microtubule-associated protein 1 light chain 3 (LC3)-II. No significant differences in spontaneous autophagy were found between T lymphocytes from patients with SLE and from healthy donors, apart from CD4+ naive T cells from patients with SLE in which constitutively higher levels of autophagy (P

Mutations in the presenilin 2 (PSEN2) gene are less commonly identified as genetic causes of early-onset familial Alzheimer's disease than mutations in the amyloid precursor protein (APP) and the presenilin 1 (PSEN1) genes. In fact, only 23 different mutations in the PSEN2 gene have been described in the literature. This paper deals with a sporadic case of a 55 year-old subject bearing an amino acid substitution from arginine to tryptophan at codon 71 of PSEN2 and presenting a peculiar early-onset Alzheimer's disease phenotype.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder leading to slow neuronal loss in several brain regions. It is characterised by the presence of cerebral senile plaques comprised of aggregated amyloid-β peptides. Transcriptional regulation of the γ-secretase complex, which cleaves the β-amyloid precursor protein to produce Aβ-peptides, could modulate the pathological phenotype of AD patients. This study investigates whether rosuvastatin, an HMG-CoA reductase inhibitor, modulates the expression of genes involved in the function of the γ-secretase complex, in a human cellular model for Aβ peptide accumulation. In particular, we analysed the effect of the statin combined with apoptotic induction. Experimental apoptosis was induced by thapsigargin treatment, a drug that depletes intracellular calcium stores via inhibition of the calcium ATPase pump. Notably, systemic calcium dysregulation accompanies almost all of the brain pathology processes observed in AD. We found differential transcriptional regulation of some γ-secretase cofactors relative to rosuvastatin treatment, in cells expressing Swedish mutant APP. Interestingly, this statin down-regulated the transcription of some enzyme cofactors, similar to treatment with thapsigargin. However, rosuvastatin neither affected the basal Aβ levels nor counteracted APP processing or Aβ over-production triggered by the thapsigargin. Our results provide evidence that rosuvastatin alters gene expression of the γ-secretase complex without affecting enzyme activity.

Branched-chain amino acids (BCAAs), valine, isoleucine, and leucine, are widely used among athletes as dietary integrators. Although the occurrence of untoward effects of BCCA supplementation, with particular regard to neurological disturbances, cannot be excluded, no specific studies have been performed so far. The aim of this work was to evaluate the effects of a diet enriched in BCAAs on the expression of oxidative stress pathway genes in the brain of C57Bl/6J mice. Animals were fed a standard or a BCAA diet for 95 days starting from postnatal day 21 until sacrifice. BCAA treatment, at doses comparable to human usage, significantly down-regulated the expression of some antioxidant genes, while up-regulating the expression of some oxygen transporters. In conclusion, it appears that BCAAs administered by diet could alter some specific oxidative stress pathways in the brain. Caution should thus be exercised in the widespread use of BCAAs as dietary integrators in sports practice.

Background/Aims: Mutations in the amyloid precursor protein gene were the first to be recognized as a cause of Alzheimer’s disease (AD). Methods: We describe 2 Italian families showing the missense mutation in exon 17 of the amyloid precursor protein gene on chromosome 21 (Val717Ile), known as London mutation. Results: In 1 family, this mutation was responsible for AD in 3 out of 7 siblings and it is also present in a fourth sibling who has only shown signs of executive dysfunction so far. Two subjects of the other family with AD diagnosis were carriers of the same mutation. Conclusion: All AD subjects showed a cognitive profile characterized by early impairment in long-term memory, shifting abilities and affective symptoms beginning in the fifth decade of life.

The metabolism of sphingolipids in the central nervous system (CNS) has been studied in adult rats by intraventricular administration of fluorescent ceramide (CER). Rats were sacrificed at various time points post inoculation and the fluorescence of CER, cerebrosides (CB), sulfatides (SULF) and sphingomyelin (SPM) was determined in the CNS myelin and in the pellet, containing mainly microsomes, obtained by Norton myelin preparation. The incorporation of fluorescence was more in the pellet than in the myelin at all times studied. Initially the fluorescence present in the pellet was prevalently due to untransformed CER but an increase of fluorescent products with time was observed. CB was the main product up to 2 h post inoculation, then it decreased with concomitant increase of fluorescent SULF. In the myelin we did not observe differences in incorporation and transformation of fluorescent CER with time: CB was the main fluorescent product at all times studied. At 0.5 h post inoculation the fluorescence, observed by fluorescence microscope, was located in the cell lining the ventricles while after 24 h it appeared also in the paraventricular areas.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Abeta peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer's disease, as APP, beta- and gamma-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of gamma-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.

Biochemical studies of myelin fractions were undertaken on Lewis rats during various time-points in the development of chronic-relapsing experimental allergic encephalomyelitis (CR-EAE). Lipid and protein composition of myelin fractions obtained by sucrose density gradient centrifugation at 10, 19, 24, and 66 d postinduction (pi) were determined by high-performance thin-layer chromatography (HPTLC) and sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS PAGE), respectively. When comparing the myelin fractions of CR-EAE affected animals with those of controls, main differences were observed at 10 d pi. These changes were particularly evident in the light myelin fraction, where a decrease in the percentage of phosphatidylethanolamine and small basic protein relative to the total lipids and proteins of the fraction were observed. At 19 and 24 d pi no biochemical differences were present in both fractions. At 66 d pi, differences in the lipid composition were observed again only in the light myelin fraction. These findings suggest that the light myelin fraction is the most sensitive, particularly at the early stages of the disease, and must play a key role in demyelinating processes.