Michael M. Zeineh, Ph.D. - US grants

DESCRIPTION (Adapted from applicant's abstract): The goal of this project is to better define the functional anatomy of the human hippocampus in memory processing with fMRI. This project proposes to perform high-resolution structural imaging studies of the hippocampal region, examining activity in the CA fields, subiculum, and entorhinal cortex throughout the anterior-posterior axis bilaterally, during performance of several memory paradigms in normal volunteers. Using computational techniques, the applicant will unfold the hippocampus to provide a flattened functional map of its activity. This research aims to elucidate the functional organization of substructures within the hippocampus in the intact brain. This knowledge will also impact the understanding and treating of diseases involving the hippocampus such as Alzheimer's disease and temporal lobe epilepsy. There will be three phases to this research: 1) The technology will be developed and optimized for the high-resolution imaging and segmentation of the hippocampus. 2) Stimulus paradigms will be developed with the goal of achieving differential hippocampal activity. 3) In order to identify structure-function relationships, these paradigms will be combined with the high-resolution imaging technology to deliver functional flattened maps of hippocampal activity.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The impact of Alzheimer's disease (AD) on the aging population of the world is growing. While there is an extensive literature on the importance and pathological evolution of amyloid plaques in AD, imaging this pathogenesis remains elusive.

Alzheimer?s  disease  (AD)  afflicts  millions  of  Americans,  but  no  effective  treatments  exist.  Although  abnormal  proteins  (amyloid  and  tau)  accumulate  and  accompany  neurodegeneration  in AD,  recent  studies  suggest  that  inflammation  led  by  the  brain?s  immune  cells  (microglia)  is  an  important  factor.  However,  inflammation is challenging to measure in living patients. Iron is a key component of inflammation and may be  toxic in AD. Because it can be measured by MRI, iron may be a practical surrogate measure for inflammation.   Coupling MRI with pathology slides has shown that iron is present within microglia in AD, in particular in a part  of  the  brain  important  for  memory,  the  hippocampus.  This  project  proposes  that  iron-­containing  microglia  accumulate  early  in  the  disease  process.  The  end  result  of  this  inflammation  could  be  tau  accumulation  and  neuronal death. If iron-­containing microglia can be detected by MRI early in the disease process, before memory  has become too impaired, this could revolutionize AD diagnosis and treatment.   The project goals are as follows. (1) Determine whether the accumulation of iron-­containing microglia is  concomitant with widespread amyloid deposition and antecedent to tau propagation. This project will examine  the  post-­mortem  hippocampus  of  patients  with  and  without  AD  pathology.  By  combining  MRI  with  a  careful  examination  of  pathology  slides,  this  project  will  prove  that  MRI  is  measuring  iron-­containing  microglia  across  the stages of AD. We will also see how the iron, microglia, amyloid, and tau overlap with one another in human  brain  specimens.  (2)  Determine  whether  iron  is  higher  in  the  hippocampus  in  AD  compared  to  no  AD  using  advanced  microscopy.  This  project  will  employ  X-­ray  microscopy  and  electron  microscopy  to  measure  exactly  how much iron is in the hippocampus, and to define the precise location where it is increased in AD. (3) Determine  whether iron-­containing microglia are present along with amyloid and tau in living AD patients, present with along  amyloid  in  patients  with  mild  or  no  AD  symptoms,  and  absent  in  patients  with  no  amyloid.  This  proposal  will  recruit patients with AD, patients with mild cognitive impairment, and healthy control participants from Stanford?s  Alzheimer?s  Disease  Research  Center.  We  will  balance  for  gender  and  age  across  groups,  perform  a  full  neuropsychological evaluation, and obtain APOE status. Participants will undergo three studies: a 7T GRE MRI,  an amyloid PET-­MR scan, and a tau PET-­MR scan. This project will show how iron-­containing microglia, amyloid,  and tau overlap with one another in living humans.  The contribution of this project will be to show that 7T GRE MR measures iron-­containing microglia. This  measurement  can  serve  as  an  effective  biomarker  for  inflammation  in  early  AD.  This  project  will  enable  many  exciting opportunities to test new therapies before the onset of memory decline. 

PROJECT SUMMARY/ABSTRACT Alzheimer?s disease (AD) afflicts millions of Americans, yet no effective treatments exist. Iron has been shown to be involved in key AD pathologic processes, including amyloid and tau aggregation, inflammation, oxidative stress, and cell death mechanisms. Despite this growing evidence, it is challenging to ascertain alterations in iron metabolism in vivo, limiting potential translation to biomarkers and novel therapies. Exosomes are nanometer-sized vesicles shed by cells to transport proteins, nucleic acids, metals, lipids or metabolites. While exosomes reflect cellular processes and can reveal disease-related pathologies in human tissues and biofluids, iron abnormalities in AD exosomes have not yet been investigated. We will address this knowledge gap through state-of-the-art exosome isolation technology combined with advanced iron imaging, protein quantification and next generation sequencing methods. Our goal is to investigate iron dysregulation in exosomes from post- mortem AD brains, in order to unveil AD-specific biomarkers and facilitate the development of novel therapies. The project aims are: (1) To determine whether the quantity, oxidation state, and cellular origin of exosomal iron is altered in AD. Using MRI and synchrotron X-ray microscopy, we will quantify tissue iron content and oxidation state in human AD and control hippocampal specimens. We will then use our novel exosome isolation platform, ExoTIC, to isolate exosomes from regions of high hippocampal iron content in the same specimens. Using antibodies that target cell-surface proteins, we will enrich the isolated exosomes based on their cellular origin (e.g. neurons, microglia, etc.). We will quantify exosomal iron content from each cell type using mass spectrometry (ICP-MS), and measure exosomal iron oxidation state using electron microscopy. Taken together, we will determine whether iron content and oxidation state are altered in Alzheimer?s exosomes compared to controls, in particular in exosomes originating in microglia, the brain?s immune cells. (2) Detect dysregulation of iron-related proteins and RNAs in AD exosomes. Using Western blotting on the enriched exosomes, we will determine whether levels of proteins that play a role in iron metabolism are altered in AD compared to controls. Because exosomes are generally rich in microRNAs that are known to regulate gene expression, we will use RNA-Seq to determine whether exosomal microRNAs regulating these same iron-related proteins are also altered in AD. Machine learning algorithms will enable the creation of an atlas of microRNAs linking iron, iron-related proteins, and neuropathology, which should provide a deeper understanding of AD biology. Characterization of exosome content in the AD brain should result in cell-specific signatures of iron dysregulation associated with neurodegeneration. This approach may elucidate novel aspects of AD biology, lead to novel assays to detect early AD, and facilitate a much-needed future therapy.