RNA analysis in neuronal dendrites: insights into Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative movement disorder that occurs following selective neuronal loss within the substantia nigra pars compacta. A number of symptoms result, including resting tremor, rigidity, akinesia and postural instability. In addition to neuronal loss, the disease is characterized by widespread Lewy body pathology, which implies the presence of intracellular Lewy bodies and dystrophic Lewy neurites. Lewy bodies are neuronal, intracytoplasmic spherical inclusions that contain an eosinophilic core surrounded by a pale halo. They are associated with coarse dystrophic neurites called Lewy neurites. Given the correlation between Lewy body pathology and neurodegeneration, much work has focused on defining the properties of these filamentous inclusions, including their biochemical composition, mechanisms of aggregation and effects on cell viability [1]. In addition to understanding Lewy body pathology, there has been an emphasis on identifying and characterizing the genes associated with rare, familial forms of PD [2,3]. These strategies have served as entrance points in understanding the pathogenesis of PD. Similar strategies are employed for the study of other neurodegenerative diseases, including Alzheimer’s disease and frontotemporal dementia.

While these strategies continue to yield insights into the pathogenesis of PD, as well as other neurodegenerative diseases, we believe they contain some significant limitations and need to be complemented with other approaches. The emphasis on rare, familial forms of the disease does allow for unobstructed analysis of clinically relevant, single gene defects. However, the vast majority of PD cases are sporadic and likely dependent upon multigenic and environmental effects. The study of inclusions and their components does identify areas of disease pathogenesis that likely impact aspects of neurodegeneration. However, a more thorough survey of the diseased cellular microenvironment is needed. Recently, there has been a greater emphasis on systems-based genomic approaches to analyze gene-expression changes in PD. A number of studies have analyzed gene expression in methylphenyltetrahydropyridine-treated mice, genetic models of PD in insects and rodents, as well as from post-mortem PD tissue [4]. The former analyze changes due to known causes of PD, with greater accessibility to different brain regions and stages of disease. The latter provide RNA from actual sporadic cases, but with limited accessibility to diverse tissue. The hope is that a consensus expression profile unique to a specific brain region and stage of disease can emerge, from which one can identify biomarkers, better understand pathogenesis and pinpoint therapeutic targets.

Much of the expression profiling in PD, as well as other diseases, has focused on utilizing groups of cells for analysis. Unfortunately, this leads to contamination from nonspecific cellular sources. It also misses the complexity in the cellular expression profile that can vary from cell to cell as a result of disease. Furthermore, few studies to date have focused on expression analysis from subcellular neuronal compartments within the context of neurodegenerative disease. The first degenerative signs of PD appear in the synapses as well as the dendrites and axons. Lewy body pathology has been shown to be present in neurites at early stages of disease. Consequently, these subcellular regions likely undergo expression changes that contain unique information relevant to disease pathogenesis. Only focused analysis of these neuronal, subcellular compartments will provide the sensitivity and specificity needed to accurately identify relevant gene-expression changes and other aspects of pathogenesis. Hence, the unbiased analysis of neuronal subcellular regions will be essential in pinpointing relevant macromolecules involved in the pathogenesis and neuronal death processes associated with neurodegenerative disease.

The ability to identify and describe changes in mRNA populations within subcellular regions, such as neuronal dendrites, has been the focus of various laboratories for a number of years. Early methodologies relied on in situ hybridization using radiolabeled probes to identify mRNA species in dendrites. These procedures were time intensive and had limited sensitivity. Consequently, only a few mRNAs were identified in dendrites. Since then, a number of methodologies have been developed that allow for the empirical identification of a larger fraction of mRNAs in dendrites from primary neuronal cultures [5]. Here, single cell dissection methods allow for the selective physical isolation of a single dendrite or groups of dendrites. Then, with the aRNA amplification methodology, mRNA populations derived from dendrites are copied in a manner that preserves relative mRNA abundances. Finally, individual mRNA species are identified by screening cDNA libraries, profiling with microarrays or by sequencing. Using these methods, over 800 mRNA species have been identified in this subcellular compartment from primary rat and mouse hippocampal neurons. They have been shown to fall within multiple functional classes, including neurotransmitter receptors, integral membrane signaling proteins, enzymes and cytoplasmic regulatory proteins, as well as nuclear transcription factors.

Identifying dendritic mRNAs from the substantia nigra and changes in their expression as a result of PD will provide important information regarding the degenerative process, as well as the cellular response to it. Analysis of dendritic compartments from downstream cellular components of the motor loop will also provide information regarding the pathogenic effects of losing substantia nigra input. Currently, technical limitations make the specific isolation of processes from post-mortem human sections challenging, especially where a clear dendritic field is lacking. The development of improved labeling techniques and single cell dissection methods for sections is needed to overcome these limitations. Culturing substantia nigra neurons from animal models and characterizing dendritic mRNA populations is an alternative. Then, through modern in situ hybridization technologies, the dendritic expression of identified mRNAs can be verified in post-mortem sections and compared between cases and controls. Another alternative approach is the utilization of synaptoneurosome fractions from post-mortem tissue. Synaptoneurosomes are fractions enriched in synaptic components from which mRNA populations can be amplified and arrayed for identification. These alternative approaches can serve complementary functions.

Dendritically localized mRNAs can be translated in the dendritic compartment [6]. Given this, the localization of mRNAs within this subcellular compartment strongly suggests that the local translation of these mRNAs is somehow unique from translation occurring in the cell soma. In response to local cues, dendritically translated proteins function locally to modulate dendritic function. This idea has been supported in studies demonstrating the importance of local protein synthesis in long-term potentiation and long-term depression [7]. In addition, dendritically translated proteins are different from their soma counterparts and, if transported to other compartments, can function differently from soma-synthesized proteins. Using a novel regionalized mRNA transfection methodology, it has been observed that dendritically synthesized Elk-1 protein uniquely leads to neuronal death following transport into the nucleus, whereas the somatically synthesized Elk-1 protein does not [8]. While these are still emerging concepts, local dendritic cues during neurodegenerative disease could alter both dendritic mRNA expression levels as well as the protein synthesis of dendritically localized mRNAs. Characterizing these effects may significantly impact our understanding of PD pathogenesis. Furthermore, it may lead to the development of therapeutics specifically targeted to subcellular compartments and regionalized cellular pathology.

Financial & competing interests disclosure

J Eberwine is a co-inventor of the aRNA procedure and consults for, and has an ownership interest in, LBS Technologies, which has licensed the technology. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.