AIM To investigate whether the changes of amyloid ß1-42, total tau (t-tau), phospho-tau (p-tau), as well as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression and their concentration in the conjunctiva, tear, nasal mucosa and saliva of patients with Alzheimer's Disease (AD) may represent useful biomarkers to the diagnosis of AD and to identify subjects at risk of progression
DESIGN 2-year prospective, observational study
METHODS Patients fulfilling diagnostic criteria for typical AD (n= 40), prodromal AD (n=40), and non-AD cognitive impairment (n=40) and cognitively normal age and sex matched controls (n=40) will be included. All subjects will undergo extensive neuropsychological examination, and magnetic resonance imaging of the brain. Lumbar puncture and cerebro-spinal fluid (CSF) evaluation will be carried out in patients only. Enrolled patients have to show presence of medial temporal lobe atrophy and abnormal cerebrospinal fluid biomarkers with low amyloid ß1-42 concentrations, increased total tau concentrations or increased phospho-tau concentrations or combinations of the three. Patients and controls will be evaluated at baseline and every 6 months for 2 years. Biological samples of tears, conjunctival and nasal cytology as well as salivary samples will be collected at each time points. Levels of Amyloid Precursor Protein and its cleavage products, amyloid ß1-42, total and phosphorylated tau protein, NGF, pro-NGF, BDNF and pro-BDNF will be assessed by specific ELISA and Western blot and values will be compared among groups and over time using analysis of variance.
Tear and conjunctival ocular biomarkers levels, as well as nasal and salivary biomarkers concentrations will be further correlated to neuropsychological scores, structural data at MRI, and CSF values by linear or logistic regression analysis. Receiving operating characteristics curves will be calculated to identify the most useful diagnostic tool for AD
Alzheimer¿s disease represents the most common cause of dementia in elderly. It affects 35.6 million of individuals with estimated costs of 818 billion USD worldwide in 2015. In addition, AD prevalence is expected to increase to 65.7 million people in 2030 and to 115.4 million in 2050 due to the anticipated increase in life expectancy.
Currently, patients with AD are diagnosed when cognitive loss has been already started. Cognitive evaluations, brain imaging and cerebrospinal fluid (CSF) evaluations represent the standard diagnostic methods for differential diagnosis with other vascular and neurological forms of dementia. However, the identification of biomarkers able to allow an early diagnosis of AD should represent a breakthrough in the management of this challenging and life-threatening condition. In fact, functional and morphological alterations associated with AD begin years, even decades, before the onset of clinical manifestations and an early diagnosis could allow a prompt intervention, before the cognitive loss, with higher success rate. (1)
Therefore, the main challenge of AD research is the discovery of reliable and predictive biomarkers useful for an early diagnosis, before the loss of autonomy and the onset of dementia. In addition, with the advent of novel treatments effective in slowing the progression of lesions in AD, the race for biomarkers¿ research is a top priority. Currently, pathological biomarkers of AD are detected by invasive and expensive procedures such as CSF analysis, brain imaging or post-mortem.
In this context, the aim of this project is to investigate whether the changes of amyloid 1¿42 (A42), total tau (t-tau), phospho-tau (p-tau), as well as NGF and BDNF expression and concentration in the conjunctiva, tear, nasal mucosa and saliva of patients with AD may represent useful biomarkers to the diagnosis of AD and/or to identify subjects at risk of progression. (1)
Since APP phosphorylation occurs before and favors amyloid plaques appearance, the advantage of using ocular surface levels of APP and/or APP cleavage products as ocular biomarker for AD is noteworthy, also when compared to other non-invasive tests, like the recently prospected imaging of the retinal amyloid burden. In fact, tear and conjunctival sampling is easy, rapid and non-invasive and provide biological samples to be easily assessed for the levels of expression of AD-specific biomarkers.
The other aim of this study is to evaluate ocular surface, nasal and salivary expression of neurotrophins as potential biomarkers of disease progression and prognosis. Among neurotrophins¿ family, alteration of Nerve Growth Factor (NGF) pathway in the pathogenesis of Alzheimer¿s disease is well established by experimental and clinical studies. NGF was discovered in the 1950s by Nobel Prize Rita
Levi-Montalcini. NGF is essential for the development and maintenance of neurons in the peripheral nervous system and for the integrity of cholinergic neurons in the central nervous system. It has been demonstrated that NGF is involved in AD pathogenesis and that impaired NGF retrograde transport is related to cholinergic neurodegeneration in AD.(14) In 2005, Mark Tuszynski et al successfully performed a phase I clinical trial based on these findings, showing that NGF gene therapy is a safe treatment and might slow or prevent the neuronal degeneration and cell death characteristic of AD. (10) Our preliminary data demonstrated a significant increase of neurotrophin¿ precursors (pro-NGF and pro-BDNF) in tears of AD patients when compared to healthy subjects, suggesting to evaluate their use as diagnostic and/or prognostic biomarkers in a prospective study. Our data might support the hypothesis that an unbalance in neurotrophins metabolism, with a relative increase of the proapoptotic form (pro-neurotrophin) may contribute to the progressive loss of cholinergic neurons. Interestingly the demonstration that pro-neurotrophins levels are increased in local tissues, such as eye and nose, of AD patients supports the hypothesis that AD cannot be considered only a ¿brain¿ disease.
Our study aims at identifying an early and yet unexplored biomarker of AD in the eye. Given the urgent need for an early and accurate AD diagnosis, the development of an easy and non-invasive test for early diagnosis of AD and/or disease progression is noteworthy. In addition, novel AD biomarkers¿ discovery may both bring advance to the clinical practice and improve the assessment of treatments efficacy in clinical trials.