Neuroscience

Dopaminergic (DAergic) neurodegeneration in the substantia nigra pars compacta of the human brain is the pathological feature associated with Parkinson’s disease (PD). Drosophila also exhibits mobility defects and diminished levels of brain dopamine on exposure to neurotoxicants mimicking PD. Our laboratory demonstrated in a Drosophila model of sporadic PD that there is no decrease in DAergic neuronal number; instead, there is a significant reduction in tyrosine hydroxylase (TH) fluorescence intensity (FI). Here, we present a sensitive assay based on the quantification of FI of the secondary antibody (ab). As the FI is directly proportional to the amount of TH synthesis, its reduction under PD conditions denotes the decrease in the TH synthesis, suggesting DAergic neuronal dysfunction. Therefore, FI quantification is a refined and sensitive method to understand the early stages of DAergic neurodegeneration. FI quantification is performed using the ZEN 2012 SP2 single-user software; a license must be acquired to utilize the imaging system to interactively control image acquisition, image processing, and analysis. This method will be of good use to biologists, as it can also be used with little modification to characterize the extent of degeneration and changes in the level of degeneration in response to drugs in different cell types. Unlike the expensive and cumbersome confocal microscopy, the present method will be an affordable option for fund-constrained neurobiology laboratories.

Key features

• Allows characterizing the incipient DAergic and other catecholaminergic neurodegeneration, even in the absence of loss of neuronal cell body.

• Great alternative for the fund-constrained neurobiology laboratories in developing countries to utilize this method in different cell types and their response to drugs/nutraceuticals.

Graphical overview

Many single nucleotide polymorphisms (SNPs) identified by genome-wide association studies exert their effects on disease risk as expression quantitative trait loci (eQTL) via allele-specific expression (ASE). While databases for probing eQTLs in tissues from normal individuals exist, one may wish to ascertain eQTLs or ASE in specific tissues or disease-states not characterized in these databases. Here, we present a protocol to assess ASE of two possible target genes (GPNMB and KLHL7) of a known genome-wide association study (GWAS) Parkinson’s disease (PD) risk locus in postmortem human brain tissue from PD and neurologically normal individuals. This was done using a sequence of RNA isolation, cDNA library generation, enrichment for transcripts of interest using customizable cDNA capture probes, paired-end RNA sequencing, and subsequent analysis. This method provides increased sensitivity relative to traditional bulk RNAseq-based and a blueprint that can be extended to the study of other genes, tissues, and disease states.

Key features

• Analysis of GPNMB allele-specific expression (ASE) in brain lysates from cognitively normal controls (NC) and Parkinson’s disease (PD) individuals.

• Builds on the ASE protocol of Mayba et al. (2014) and extends application from cells to human tissue.

• Increased sensitivity by enrichment for desired transcript via RNA CaptureSeq (Mercer et al., 2014).

• Optimized for human brain lysates from cingulate gyrus, caudate nucleus, and cerebellum.

Graphical overview

Missense mutations in leucine rich-repeat kinase 2 (LRRK2) cause forms of familial Parkinson’s disease and have been linked to ‘idiopathic’ Parkinson’s disease. Assessment of LRRK2 kinase activity has been very challenging due to its size, complex structure, and relatively low abundance. A standard in the field to assess LRRK2 kinase activity is to measure the level of substrate phosphorylation (pThr73-Rab10) or autophosphorylation of serine 1292 (i.e., phosphoserine 1292; pS1292). The levels of pS1292 have typically been assessed by western blotting, which limits cellular and anatomical resolution. Here, we describe the method for a novel proximity ligation assay (PLA) that can detect endogenous LRRK2 kinase activity (PLA LRRK2) in situ at cellular and subcellular resolutions. PLA is a fluorescence- or chromogen-based assay that can be used to either (1) detect protein-protein interactions or (2) detect and amplify post-translational modifications on proteins. We used PLA for in situ detection and amplification of LRRK2 autophosphorylation levels at serine 1292. Our findings demonstrate that PLA LRRK2 is a highly sensitive and specific assay that can be used for assessing kinase activity in cultured cells and postmortem tissues.

Parkinson’s disease is a progressive neurodegenerative movement disorder that happens due to the loss of dopaminergic neurons in the substantia nigra. The deficiency of dopamine in the basal nuclei drives cardinal motor symptoms such as bradykinesia and hypokinesia. The current protocol describes the cylinder test, which is a relatively simple behavioral assessment that evaluates the motor deficits upon unilateral degeneration of the nigrostriatal pathway in experimental models of Parkinson’s disease. Since dopamine-depleted mice exhibit the preferential use of the forelimb ipsilateral to the lesion, here researchers perform the cylinder test to investigate the therapeutic effects of antiparkinsonian treatments on the performance of the contralateral (injured) limb.

Studying the aggregation of amyloid proteins like α-synuclein in vitro is a convenient and popular tool to gain kinetic insights into aggregation as well as to study factors (e.g., aggregation inhibitors) that influence it. These aggregation assays typically make use of the fluorescence dye Thioflavin T as a sensitive fluorescence reporter of amyloid fibril formation and are conducted in a plate-reader-based format, permitting the simultaneous screening of multiple samples and conditions. However, aggregation assays are generally prone to poor reproducibility due to the stochastic nature of fibril nucleation and the multiplicity of modulating factors. Here we present a simple and reproducible protocol to study the aggregation of α-synuclein in a plate-reader based assay.