Advanced Tissue Resource Center (ATRC)

Overview

The research efforts at the Advanced Tissue Resource Center, part of the Department of Neurology at Mass General, have led to significant contributions to the understanding of many types of neurologic and psychiatric diseases.

We have successfully employed the highly specialized technological process of laser capture microdissection to determine which specific molecules are contained in individual cells and/or regions of the brain.

Research Areas

Historically, we have participated as members of neuroscience teams studying the following:

• Alzheimer’s disease
• Amyotrophic lateral sclerosis (ALS)
• Autism
• Bipolar disorder
• CADASIL (cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy)
• Huntington’s disease
• Multiple sclerosis
• Muscle atrophy
• Parkinson’s disease
• Post-traumatic stress disorder (PTSD)
• Schizophrenia
• Stroke
• Synucleinopathies
• Tauopathies
• Traumatic brain injury (TBI)

Ongoing Research

Our ongoing research projects focus on several areas of basic and clinical neuroscience, including:

• The profiling of microRNAs (miRNA) and other non-coding ribonucleic acids (RNAs) in neurodegenerative diseases
• Exosome-associated biomarkers of neurologic disease
• The development of single-cell expression profiling methodologies via laser-capture micro-dissection and digital droplet polymerase chain reactions (PCR)
• Fiber type-specific gene and protein expression of atrophic skeletal muscle
• The role of matricellular proteins in neurodegeneration and neuroinflammation

Research Projects

Here are several of the ongoing research projects at the ATRC.

Single-Cell Expression Profiling via Laser Microdissection and Digital Droplet Polymerase Chain Reaction (PCR)

The objective of this study is to profile microRNAs in brain tissue and biofluids of patients with various neurodegenerative and psychiatric diseases.

MicroRNAs (miRNA) are short, non-coding ribonucleic acids (RNAs) that control gene expression at the posttranscriptional level by binding to the 3' untranslated region of target messenger RNAs (mRNAs), causing their translational inhibition or degradation.

Each miRNA can regulate thousands of different mRNA targets, modifying an exceptionally large number of genes, whereas a single mRNA is translated into a single protein. Ergo, miRNA levels may provide a better indication of a cell’s physiological state in health and in disease.

A demonstration of the cell-selective accuracy of laser-capture microscopy (LCM). Shown are microdissections of human brain tissue down to the single cell level, allowing us to isolate individual neural, glial and vascular components. The upper panels show intact, appropriately immunostained brain tissue sections indicating the cellular targets (astrocytes, capillary endothelial cells, neurons or microglia) while the lower panels show the corresponding post-LCM cellular target microdissected onto the surface of a collection device.

Identifying miRNA expression signatures has thus become an attractive technique that can produce robust neurophysiological and neuropathological data. We and others have begun to assess the levels of microRNAs in brain tissue, blood and cerebrospinal fluid (CSF) of patients with various neurodegenerative and psychiatric diseases.

The importance of non-coding RNA based regulation has become even more apparent of late. A recent Cell review entitled “A ceRNA Hypothesis: The Rosetta Stone of a Hidden RNA Language” describes how non-coding RNAs (ncRNAs) may “talk” to each other in a unified way, hypothetically predicting a previously unimagined ncRNA-dependant regulatory network.

Also appearing recently is a report (Cell Research (2012) 22:107–126) that rice-specific miRNA 168a can transfer to the digestive system, circulating blood and the liver of mammals upon consumption, and directly regulate the low density lipoprotein receptor adaptor protein. This extraordinary finding implies that miRNA signaling is so important and conserved that its effects can even be transferred across kingdoms.

As such, miRNA’s role as signaling moieties (the signaling parts of a molecule) and master regulators of cellular processes is affirmed, making them excellent candidates for biomarkers of physiologic and pathologic states. miRNAs are also more stable and resistant to degradation and posttranslational modification than mRNAs and proteins, making them better candidates for biomarker studies. Additionally, miRNAs can be amplified from samples with rarified content, whereas proteins cannot.

By using sensitive and specific quantitative reserve-transcriptase polymerase chain reaction (qRT-PCR) technology, it is possible to detect altered expression of regional and stage-specific miRNAs in the brains and biofluids of these patients. Many of the miRNAs have been linked to molecular pathways, including neuronal differentiation, glutamate metabolism, innate immunity and cell survival.

For example, miR-9 and miR-132 downregulation has been correlated to impaired neurogenesis and neuronal differentiation. These miRNA profile changes might be specific markers for sporadic Alzheimer’s disease (AD). In fact, any miRNA changes could be disease-causing or may exacerbate an underlying pathology.

Thus, linking miRNAs to their specific targets could reveal novel pathways for disease progression. By analogy, if any neurodegenerative disease is caused or exacerbated by altered miRNA(s), then these could potentially serve as sensitive and specific biomarkers and thereby reveal the underlying disease pathology.

However, we have come to realize that miRNA expression changes in brain tissue occur at relatively modest levels. Subtle fold-changes on the order of <1 to ~2 fold are enough to alter the balance of a cell’s expression of a given miRNA-regulated protein or ontological cassette. Therefore, detecting and accurately documenting such small changes in neuropathological samples presents a considerable technical challenge.

We have taken an approach which couples the cell-selective accuracy of laser-capture microscopy with the distinct assay sensitivity advantage of digital droplet PCR (ddPCR). Thanks to Poisson mathematics, no housekeeper miRNAs or DDCt calculations are needed for ddPCR, and the very small input requirement allows us to place the entire laser-captured cell contents into a single 20,000 nano-droplet ddPCR reaction. This allows us to assay individual neural cells using a multiplexed HEX/EVA assay in order to count the molecular numbers of candidate miRNA(s) and accurately assess comparative expression ratios.

Currently, we are building a repository of miRNA expression values in the various neural cellular components of non-diseased control samples.

We are looking forward to using this technique on various neurodegenerative disease samples for comparison.

SPARC/Hevin Modulation in Alzheimer's Disease-related Cell Injury

The objective of this study is to confirm the participation of Secreted Protein Acidic and Rich in Cysteine (SPARC) and hevin in the neuroinflammatory processes associated with Alzheimer’s disease.

Alzheimer’s disease is an age-related progressive form of dementia that features neuronal loss caused by intracellular and extracellular protein deposition. Neurodegeneration is accompanied by neuroinflammation that mainly involves microglia, the resident innate immune cell population of the brain.

During Alzheimer’s disease microglia shift their phenotype, and it has been suggested that they express matricellular proteins (in particular SPARC and hevin) that facilitate migration of other immune cells, such as blood-derived dendritic cells. These proteins contribute to the neuroimmune response and play a crucial role in functional tissue repair.

By investigating post-mortem human brain tissue, we confirmed the participation of SPARC and hevin in neuroinflammatory processes and suggest an infiltration of myeloid-derived immune cells to the areas of diseased tissue. SPARC is highly expressed during Alzheimer’s disease and collocates to protein deposits, thus conducing actively to cerebral inflammation and subsequent tissue repair.

Fiber Type-Specific Gene Expression of Metachromatically Stained Skeletal Muscle

The objective of this study is to demonstrate the importance of fiber type-specific expression profiling in understanding skeletal muscle biology, and provides a practical method of performing such research.

Skeletal muscle contains various myofiber types closely associated with satellite stem cells, vasculature and neurons. During injury, growth, atrophy or diseases of skeletal muscle, the myofiber types, ratios of these types within a given muscle, and recruitment/loss of myofibers from motor units change, which makes it necessary to view skeletal muscle not as a whole but rather as a collection of microscopic myofiber sub-populations.

Thus, when performing genetic or proteomic expression analysis in any of the neuromuscular diseases, it is difficult to obtain sufficient cellular specificity to resolve differences associated with individual myofiber or motor unit sub-types.

We are using a simple histochemical method (metachromatic staining) to simultaneously identify Type I, IIA, IIB and IIC myofibers in frozen muscle sections of control and atrophic muscle followed by laser-capture micro dissection (LCM) of individual fiber types, myonuclear domains and satellite cells. Quantitative real time polymerase Chain Reaction (qPCR) was used to verify the integrity of the cell-specific RNAs harvested, while qPCR and surface enhanced laser desorption ionization-time of flight mass spectromer (SELDI-TOFMS) were used to quantify atrophy-associated changes in mRNA and protein levels within individual fiber types that could not be resolved by expression analysis of whole muscle.

Analysis of Exosomal miRNA Biomarkers of Cerebrospinal Fluid in Alzheimer's Disease Patients

The objective of this study is to test the hypothesis that the exosomal miRNA content of the cerebrospinal fluid (CSF) of humans with Alzheimer's disease differs from the content of non-demented controls.

Alzheimer’s disease (AD), the most common type of aging-associated dementia, is characterized by progressive accumulation of intracellular neurofibrillary tangles that are composed of aggregates of abnormal hyperphosphorylated microtubule-associated protein tau, and extracellular amyloid plaques comprised of amyloid β-peptide (Aβ), which are produced by processing of amyloid precursor protein (APP).

Human CSF-derived exosomes purified by differential ultracentrifugation and prepared for electron microscopy show 50-60nm diameters. 30,000x magnification.

Plaques and tangles are accompanied by neuron degeneration and predominantly affect regions of the basal forebrain, hippocampus and association cortices.

To date, the only available treatments have modest effects on disease progression and on relieving symptoms,possibly due to delayed diagnosis and treatment initiation.

AD-related changes in the brain are well advanced by the time the symptoms of dementia appear. Thus, understanding the early pathogenesis of the disease and discovering a reliable early diagnostic test or biomarker for AD before the clinical symptoms occur might increase the likelihood to attenuate AD progression before there is a significant neuronal loss.

Recent evidence suggests that microRNAs (miRNAs), short non-coding RNA molecules that regulate gene expression, may play a critical role in AD, and that such miRNAs are present in exosomes (organelles in cells) and can be used as diagnostic markers.

Our ongoing study shows the presence of specific miRNAs in human CSF and differences in the CSF exosomal miRNA content between AD patients and non-demented controls. The profile of these AD-related CSF exosomal miRNAs could potentially be used to establish novel diagnostic biomarkers.

Publications

View our complete list of publications at NIH.gov.