Abstract

   Analyses of gene expression in cells affected by neurodegenerative
   disease can provide important insights into disease mechanisms and
   relevant stress response pathways. Major symptoms in Parkinson’s
   disease (PD) are caused by the degeneration of midbrain dopamine (mDA)
   neurons within the substantia nigra. Here we isolated
   neuromelanin-positive dopamine neurons by laser capture microdissection
   from post-mortem human substantia nigra samples recovered at both early
   and advanced stages of PD. Neuromelanin-positive cells were also
   isolated from individuals with incidental Lewy body disease (ILBD) and
   from aged-matched controls. Isolated mDA neurons were subjected to
   genome-wide gene expression analysis by mRNA sequencing. The analysis
   identified hundreds of dysregulated genes in PD. Results showed that
   mostly non-overlapping genes were differentially expressed in ILBD,
   subjects who were early after diagnosis (less than five years) and
   those autopsied at more advanced stages of disease (over five years
   since diagnosis). The identity of differentially expressed genes
   suggested that more resilient, stably surviving DA neurons were
   enriched in samples from advanced stages of disease, either as a
   consequence of positive selection of a less vulnerable long-term
   surviving mDA neuron subtype or due to up-regulation of neuroprotective
   gene products.

   Keywords: Parkinson’s disease, gene expression, RNA sequencing, disease
   duration, neurodegenerative disease, cell death, neuroprotection

Introduction

   Parkinson’s disease (PD) is the most common neurodegenerative motor
   disorder, affecting 1−2% of individuals over 65 years. A hallmark of
   the disease is the progressive degeneration of midbrain dopamine (mDA)
   neurons in the substantia nigra pars compacta (SNc) and the
   accumulation of intraneuronal Lewy bodies containing misfolded
   α-synuclein in brainstem and neocortex ([39]Damier et al., 1999). In
   particular during early stages of disease, motor functions are affected
   and major symptoms include bradykinesia, rigidity, tremor and postural
   instability. Parkinson’s disease patients also suffer from non-motor
   symptoms such as depression, olfactory dysfunction, sleep disorders,
   hallucinations and cognitive impairments ([40]Chaudhuri and Schapira,
   2009). Other diseases are also associated with Lewy bodies, including
   dementia with Lewy bodies. In addition, Lewy bodies are often found in
   aged brains at autopsy also from individuals lacking symptoms. Such
   cases are often referred to as incidental Lewy body disease (ILBD). It
   remains unknown if ILBD represents a pre-clinical stage PD or if it is
   a unique pathological abnormality but as ILBD has been shown to have
   nigrostriatal dopaminergic losses midway between normal and PD
   subjects, it remains a likely candidate for a preclinical PD stage
   ([41]Gibb and Lees, 1988; [42]Beach et al., 2008; [43]DelleDonne et
   al., 2008; [44]Dickson et al., 2008; [45]Adler et al., 2010; [46]Iacono
   et al., 2015).

   Currently used PD treatments alleviate motor dysfunction by mDA
   signaling compensation, usually by administration of levodopa or other
   drugs that increase dopamine signaling. However, side effects develop
   over time and the treatment usually becomes progressively ineffective.
   Importantly, treatments increasing dopamine signaling does not slow
   neurodegeneration. A better understanding of the neurodegenerative
   mechanisms underlying the disease may result in identification of new
   molecular targets and permit the development of more effective
   therapies ([47]Armstrong and Okun, 2020).

   Comprehensive histopathological characterization of postmortem tissue
   of a large sample of PD cases of different disease duration suggests
   that both the soma and the axon of mDA neurons is affected early after
   diagnosis, with loss of mDA neuron markers both in the SNc and in the
   striatum. Degeneration of mDA neurons occurs rapidly within the first 4
   years post diagnosis and seems to progress rather slowly after the
   initial cellular decline ([48]Kordower et al., 2013). Moreover, despite
   the fact that idiopathic PD has a rather limited genetic component,
   studies of familial PD-linked genes have resulted in significant
   advances in the understanding of the molecular pathways implicated in
   PD. Thus, SN mDA neuron pathology has been associated with
   mitochondrial dysfunction, oxidative stress, excitotoxicity,
   inflammation and abnormal protein handling and folding ([49]Johnson et
   al., 2019).

   In addition to histopathological and genetic analyses, another tool
   that can provide insights into the pathology of PD is the analysis of
   changes in global gene expression patterns in samples from post-mortem
   PD brains. Gene expression profiling experiments have previously been
   performed on mRNA extracted from homogenates of the SN. Since mDA
   neurons only constitute a subpopulation of neurons and glial cells
   within the dissected SN such samples contain relatively few mDA neurons
   in relation to other cell types. Moreover, post-mortem samples are
   usually derived after prolonged disease when only few mDA neurons
   remain ([50]Kordower et al., 2013). Thus, mDA neurons are severely
   underrepresented in such samples. To avoid influence from other cell
   types in the analysis, mDA neurons can be isolated directly by
   laser-capture microdissection (LCM). Several previous studies have used
   this or other methods to isolate mRNA from the SN and used mRNA for
   analyses in hybridization chip arrays for detection of genome-wide gene
   expression ([51]Cantuti-Castelvetri et al., 2007; [52]Bossers et al.,
   2009; [53]Simunovic et al., 2009; [54]Zheng et al., 2010; [55]Elstner
   et al., 2011; [56]Riley et al., 2014). In another study, LCM was used
   to specifically quantify the expression of SNCA (α-synuclein) mRNA
   ([57]Gründemann et al., 2008). However, direct measurement mRNA
   expression by means of massively parallel RNA sequencing (RNAseq)
   offers a more sensitive analysis, improved dynamic range and with less
   technical variability as compared to different chip array platforms
   ([58]Nichterwitz et al., 2016).

   Tissue sampled from PD patients in previous studies were usually
   diagnosed many years before tissue collection and reports on how gene
   expression is affected during disease progression is lacking.
   Importantly, histopathological analysis of disease duration has
   demonstrated a profound loss of mDA neurons in the SN during the first
   years after diagnosis with nearly negligible loss thereafter
   ([59]Kordower et al., 2013). Thus, it can be assumed that previous
   studies have mostly analyzed gene expression in mDA neurons that are
   stably maintained after the initial rapid phase of mDA neuron decline.

   In this study, we used RNAseq to analyze genome-wide gene expression in
   mDA neurons isolated by LCM from postmortem cases of PD. Patients’
   samples are derived both early after diagnosis and after prolonged
   disease duration. Cases of ILBD and age-matched controls were also
   included in the analysis. We observed distinct transcriptional profiles
   corresponding to the differential duration of PD and we show that the
   transcriptional changes in ILBD are distinct from those seen in PD.

Materials and Methods

Samples

   Fresh snap-frozen human post-mortem tissue was provided by the Banner
   Sun Health Research Institute Brain and Body Donation Program of Sun
   City, Arizona, United States, where all human subjects donate after
   signed informed written consent [Western Institutional Review Committee
   (WIRB), Seattle, Washington] ([60]Beach et al., 2015). Sample data used
   in the preparation of this article were obtained from the Arizona Study
   of Aging and Neurodegenerative Disorders (AZSAND) database.^[61]1
   Detailed information about the samples analyzed is provided in
   [62]Supplementary Figure 1. Controls (n = 10) didn’t have any
   neurodegenerative disease and were selected to match the age and the
   gender of the PD cases. Since loss of dopaminergic markers occurs
   rapidly and is completed by 4 years post-diagnosis ([63]Kordower et
   al., 2013), we selected two groups of PD cases: early post-diagnosis
   2-4 years (n = 6) and late post-diagnosis 5-24 years (n = 6). ILBD
   cases (n = 7) were also included. Only samples with a post-mortem index
   < 5h were included. To get robust results, we used ≥six cases per
   condition ([64]Schurch et al., 2016).

Laser Capture Microdissection

   Cryosectioning was used to obtain 10 μm thick sections of the ventral
   midbrain, which were mounted on membrane glass slides (Zeiss,
   415190-9041-000) that were kept at −20∘C during sectioning and
   subsequently stored at −80∘C. Prior to LCM, the tissue was dehydrated
   by a series of ethanol washes (75% 30 s, 95% 30 s, 100% 30 s). The
   Leica LMD7000 system was used to capture NM+ neurons from the SNc. 10
   controls, 12 PD and 7 individuals with ILBD were analyzed. Cells were
   cut at ×40 magnification with a minimum laser power and collected into
   lysis buffer ([65]Picelli et al., 2013). From each SNc, 4–12 pooled
   samples of 100–200 micro-dissected cells were collected. As a
   reference, 18 samples were also collected from the cortex of control
   brains to provide a non-DA source of RNA. Cortex neurons were
   visualized using a quick Histogene (Arcturus) staining.

cDNA Library Preparation and Sequencing

   Libraries for RNA-seq were generated using the Smartseq2 protocol
   ([66]Picelli et al., 2013). cDNA libraries were tagmented using
   home-made Tn5 enzyme ([67]Picelli et al., 2014) and Nextera dual
   indexes (Illumina). The cDNA quality was checked on high-sensitivity
   DNA chip (Agilent Bioanalyzer). Illumina HiSeq 2000 was used for
   sequencing, giving 43 base pair reads after de-multiplexing.

Read Alignment and Quality Control

   Following de-multiplexing, reads were aligned to the hg19 human genome
   using STAR v2.3.0 and filtered for uniquely mapped reads ([68]Dobin et
   al., 2013). Gene expression was calculated as reads per kilobase gene
   model and million mappable reads (RPKMs) for each transcript in Ensembl
   release 69 using rpkmforgenes ([69]Ramsköld et al., 2009). The
   low-quality libraries were filtered out based on following criterias:
   <10.3% uniquely mapping reads, >68% fraction mismatches, <10.5% exon
   mapping reads, >57% 3′mapping, < 26% of all genes detected, <100000
   normalization reads. 270 samples passed the quality control.

Gene Expression and Pathway Analysis

   Principle Component Analysis (PCA) was used to analyze how individual
   samples relate to each other on overall gene expression profiles and
   DESeq2 package ([70]Love et al., 2014) to determine the differentially
   expressed genes between sets of samples.

   Pathway enrichment analysis was performed using the Toppgene suite
   ([71]Chen et al., 2009) on differentially expressed genes detected with
   DESeq2 that fulfilled the following criteria: padj < 0.05, mean RPKM
   value in control samples > 1 and 1.5 fold change.

Results

Tissue Preparation, Neuron Collection and Library Preparation

   Snap-frozen tissue blocks, received from the Banner Sun Health Research
   Institute Brain and Body donation program, were used to obtain
   cryo-sections of the ventral midbrain. Laser capture microdissection
   (LCM) was used to isolate mDA neurons from postmortem cases of sporadic
   PD, ILBD and age-matched controls ([72]Figure 1A and [73]Supplementary
   Figure 1). mDA neurons were identified by the presence of neuromelanin
   (NM) and dissected using LCM ([74]Figure 1B). Several replicates of
   100–200 individually isolated mDA neurons per individual (either PD,
   ILBD or age-matched controls) were collected. Cortical neurons from
   control brains (n = 18 pools), visualized by Histogene quick staining,
   were also collected by LCM in order to provide a reference source of
   mRNA from a brain region devoid of mDA neurons. Libraries for RNA
   sequencing were generated by the Smart-seq2 protocol ([75]Picelli et
   al., 2013). [76]Figure 1C summarizes cases used in the study and the
   number of analyzed samples per case, while [77]Supplementary Figure 1
   provides more information on all cases included in the study.

FIGURE 1.

   [78]FIGURE 1
   [79]Open in a new tab

   LCM and RNA-seq analysis of mDA neurons from postmortem cases of
   Parkinson’s disease, incidental Lewy body disease and age-matched
   controls. (A) Schematic overview of experimental design. Midbrain
   sections were used to isolate neuromelanin positive DA neurons by LCM
   and processed for RNA-seq. (B) Micrographs of ventral midbrain
   neuromelanin positive cells before and after LCM. (C) Parkinson’s
   disease, incidental Lewy body disease and control samples analyzed in
   the study.

   Libraries were sequenced (see Methods for details) and analyzed by
   various bioinformatic methods. The Spearman coefficient between
   biological replicates of neuromelanin-positive (NM+) cell samples
   averaged 0.78 and was not below 0.57 for any sample, revealing a very
   high level of overall correlation ([80]Supplementary Figure 2A).
   Principal component analysis (PCA) based on all variable genes showed
   that NM+ samples from healthy controls clustered separately from cortex
   samples along the second principal component (PC2) ([81]Figure 2A).
   Further comparison of cortex and NM+ samples revealed that 8845 genes
   were differentially expressed in the two populations (padj < 0.05, RPKM
   in controls > 1, fold change > 1.5; [82]Figure 2B and [83]Supplementary
   Table 1). Known dopaminergic markers such as TH, SLC6A3, EN1 and PITX3
   were identified among top-ranked genes enriched in NM+ samples
   demonstrating that these samples provide a good representation of the
   transcriptome of human mDA neurons ([84]Figures 2B–E). Moreover, the
   comparison demonstrated that pan-neuronal markers such as GAP43 and
   MAP2 were enriched in the NM+ cells ([85]Figure 2C) while glial cell
   markers were identified in cortex samples where dissection did not
   discriminate between neurons and other cell types including astrocytes
   and oligodendrocytes ([86]Supplementary Figure 2B). This difference was
   also apparent from gene ontology analysis ([87]Supplementary Figure
   2C).

FIGURE 2.

   [88]FIGURE 2
   [89]Open in a new tab

   RNA-seq analysis of neuromelanin positive cells in comparison to
   cortex. (A) Two dimensional PCA (PC1, PC2) of NM+ neurons (252 samples)
   and cortical neurons (18 samples). Samples are color-coded by cell
   type. (B) Volcano plot showing fold changes of individual genes in the
   DESeq2 analysis. Red: higher expression in NM+ samples (right side of
   the plot) or cortex samples (left side of the plot), (padj < 0.05, RPKM
   > 1, fold change > 1.5), Black: no significant difference. (C–E)
   Expression level per cell type for indicated genes in log[2](RPKM+1) in
   y-axes. Genes represent markers for pan-neuronal cells (C), dopamine
   neuron (D), and dopamine transcription factors (E).

Differential Gene Expression in Parkinson’s Disease Samples

   Interestingly, samples visualized by PCA showed that ILBD and control
   samples clustered mostly together while a large proportion of PD
   samples clustered separately along one of the principle components
   (PC5; [90]Figures 3A,B). Along other PCs, separation was not evident
   ([91]Supplementary Figures 3A,B). Thus, the analysis detected gene
   expression profiles that differed in PD samples as compared to controls
   and ILBD samples. In addition, disease duration affected the
   transcriptional profile of NM+ cells from PD samples along PC2 as
   evident from different clustering when comparing samples derived from
   patients with a disease duration of 2-4 years (PDearly) with those with
   disease duration of 5 to 24 years (PDlate); ([92]Figures 3C,D). We next
   considered how additional parameters affected the transcriptional
   profile of PD samples and found that samples did not cluster separately
   across any principal component based on either gender, post-mortem
   intervals (PMI; < 2.5 h versus > 2.5 h) or patient age (< 80 years
   versus > 80 years) (not shown); however, consistent with the observed
   different clustering of PDearly vs. PDlate samples, a trend for
   separation along PC2 was also seen when comparing Braak stages (I, II
   versus III, IV; [93]Supplementary Figure 3E). Thus, disease duration
   appears to have an influence on transcriptional profiles in mDA neurons
   remaining in PD patients at the time of death.

FIGURE 3.

   [94]FIGURE 3
   [95]Open in a new tab

   Segregation of Parkinson’s disease samples influenced by disease
   progression. (A) Two dimensional PCA (PC2, PC5) of Control, Parkinson’s
   disease and incidental Lewy body disease samples. PC5 separates
   Parkinson’s disease from Control and incidental Lewy body disease
   samples. Samples are color-coded by disease. (B,C) Two dimensional PCA
   (PC4, PC5) of Control and Parkinson’s disease samples. Samples are
   color-coded by disease (B) and disease duration (C). PC5 separates
   Parkinson’s disease from Control samples. (D) Two dimensional PCA (PC2,
   PC3) of PDearly and PDlate samples. PC2 separates PDlate from PDearly
   samples. Samples are color-coded by disease duration.

   Consistent with PCA demonstrating differences between control and PD
   samples, DESeq2 statistical analysis ([96]Love et al., 2014) identified
   hundreds of differentially expressed genes. In PDearly samples, 220
   genes were expressed at a higher level and 1029 genes were expressed at
   a lower level compared to healthy controls ([97]Supplementary Table 2);
   in PDlate samples, 448 genes were more highly expressed and 565 genes
   were expressed at a lower level ([98]Supplementary Table 3); in ILBD
   samples, 277 genes were more highly expressed and 397 genes were
   expressed at a lower level ([99]Supplementary Table 4), (padj < 0.05,
   RPKM in controls > 1, fold change > 1.5; [100]Figure 4A). [101]Figure
   4B displays a heat map visualizing selected top enriched genes in NM+
   neurons from PDearly (orange) and PDlate (red) and [102]Supplementary
   Figure 4B from ILBD. Top downregulated genes are shown in
   [103]Supplementary Figure 5.

FIGURE 4.

   [104]FIGURE 4
   [105]Open in a new tab

   Gene expression changes in PDearly, PDlate and incidental Lewy body
   disease. (A) Number of up- and down-regulated genes in PDearly, PDlate
   and incidental Lewy body disease (padj < 0.05, RPKM > 1, fold change >
   1.5). (B) Heatmap visualizing expression of top-upregulated genes in
   PDearly and PDlate. Sample types are indicated by color coding. Genes
   upregulated early are in the top part of the heatmap (light gray) while
   genes upregulated late are in the bottom part (dark gray).

   We focused on the top 100 up- or down-regulated genes (padj < 0.05,
   RPKM in controls > 1, fold change > 1.5) in PDearly, PDlate and ILBD
   samples and searched literature in order to identify genes that have
   been previously associated to PD. A striking observation in PDlate
   samples was the higher expression of several genes shown previously to
   confer neuroprotection, such as the pituitary adenylate cyclase
   activating polypeptide ADCYAP1 ([106]Reglõdi et al., 2004) the
   calcium-binding protein S100A4 ([107]Pedersen et al., 2004), the
   transcription factor NFIL3 ([108]Tamai et al., 2014), the regulator of
   G-protein signaling RGS10 ([109]Tansey and Goldberg, 2010).
   Neuroprotective gene such as PRDX5 ([110]Plaisant et al., 2003) was
   also expressed at a higher level in PDearly cases suggesting that some
   neuroprotective mechanisms are quite active also during early stages of
   the disease. Genes previously shown to be more highly expressed in SNc
   from PD patients, including DDIT4 ([111]Malagelada et al., 2006) and
   NPTX2 ([112]Moran et al., 2008), were also found to be enriched in
   PDlate cases. Of interest is also that variants of several regulated
   genes in both PDearly and PDlate samples, including GSTT1 ([113]Wang et
   al., 2014), CXCR4 ([114]Bonham et al., 2018), BAG3 ([115]Cao et al.,
   2017) and HLA-DRB1 ([116]Hollenbach et al., 2019), were previously
   associated with PD.

   Relatively few genes expressed at lower levels in PD samples were found
   to be associated with PD. However, LGALS3 (Galectin-3), a gene linked
   to neuroinflammation, was expressed at a lower level in PDearly samples
   and a BCL2 antagonist, was also expressed at lower levels in PDearly
   samples [see e.g., ([117]Bernardini et al., 2019)]. BAK1, the gene
   encoding a presumptive target of the E3 ubiquitin ligase Parkin, was
   expressed at lower levels in PDlate samples. Notably, quite many genes
   behaved in an opposite manner being enriched in PDearly samples but
   expressed at lower levels in PDlate samples. This further underscores
   the different profiles of gene expression seen in samples derived from
   patients at either early or late stages of disease ([118]Supplementary
   Tables 2, [119]3).

   We next focused on known mDA neuron markers and genes that cause
   familial PD (padj < 0.05, RPKM in controls > 1). The expression of
   known mDA neuron markers RET, GFRA1, LMX1B, and FOXA2 were not
   significantly affected in either PD group. Expression of DDC and LMX1A
   were the only genes that were significantly increased in PDearly but
   not in PDlate samples ([120]Figure 5A). SLC18A2, NR4A2, EN2, and OTX2
   were expressed at lower level only in PDearly, while TH, SLC6A3, DRD2,
   EN1, and PITX3 were low only in PDlate samples ([121]Figure 5A). SOX6,
   encoding a transcription factor specifically expressed in SNc but not
   in ventral tegmental area (VTA), was previously shown to be expressed
   at lower levels in NM+ cells in PD patients ([122]Panman et al., 2014).
   Consistent with this result SOX6 was the only analyzed mDA neuron
   marker that was significantly expressed at lower level in both PDearly
   and PDlate cases ([123]Figure 5A). Most genes causing familial forms of
   PD did not show differential expression in any PD cohort as compared to
   controls. UCHL1 was significantly enriched only in PDearly cases,
   ATP13A2 was enriched in PDearly but lower than controls in PDlate
   cases, while GIGYF2, EIF4G1, and PLA2G6 were lower in PDlate but not in
   PDearly cases. PINK1 and NOTCH2NL were the only familial PD-associated
   genes that was expressed at lower levels in both early and late PD
   cases ([124]Figure 5B).

FIGURE 5.

   [125]FIGURE 5
   [126]Open in a new tab

   Dopamine related gene expression changes in PDearly, PDlate and
   incidental Lewy body disease. (A–C) Arrows indicate significant (padj <
   0.05, RPKM in controls > 1) up- or down-regulation of typical DA
   markers (A), PD-associated genes (B) and SNC/VTA genes (C) in PDearly,
   PDlate and incidental Lewy body disease samples. Hyphen indicates
   nonsignificant change.

   The diversity of mDA neurons within the ventral midbrain is
   substantial. In PD, mDA neurons of the ventral-lateral SNc are more
   vulnerable than mDA neurons localized at a dorsal-medial region of the
   SNc and within the VTA ([127]Gibb and Lees, 1991). Although tissue
   samples were dissected from the substantia nigra it is likely that NM+
   cells isolated by LCM in this study represents mDA neurons belonging to
   different subtypes and with variable vulnerability ([128]Poulin et al.,
   2020). A recent study identified markers specific for human SNc and VTA
   mDA neurons, respectively (bioRxiv 334417; doi:
   [129]https://doi.org/10.1101/334417). The study defined 33 markers that
   can distinguish between human VTA and SNc mDA neurons. We assessed if
   any of these genes were enriched in either PDearly, PDlate or
   incidental Lewy body disease. Interestingly, a clear trend was observed
   when comparing the different samples ([130]Figure 5C). Accordingly, the
   majority of VTA markers were significantly enriched in PDlate samples
   but not in PDearly samples indicating that surviving cells isolated
   from PDlate samples are enriched for cells with VTA or “VTA-like” gene
   expression profiles. In contrast, although loss of neurons within the
   SNc has been reported in ILBD (40%), VTA markers were not enriched in
   ILBD samples ([131]Iacono et al., 2015) ([132]Figure 5C).

Overlap of Differential Gene Expression in Parkinson’s Disease and Incidental
Lewy Body Disease Samples

   The overlap between differentially expressed genes in PD and ILBD
   samples was limited ([133]Supplementary Figure 4A). Of note, among
   genes that are differentially expressed in both PD and ILBD samples,
   several are among those that previously have been associated with PD.
   Thus, GPNMB, PRDX5, GSTT1, HLA-DRB1, and DDIT4 were enriched in both
   ILBD and PD samples and linked to PD as described above. Moreover,
   LGALS3, a gene associated with neuroinflammation, was expressed at
   reduced levels in both PD and ILBD samples.

Gene Ontology Terms Enriched in Parkinson’s Disease and Incidental Lewy Body
Disease

   Gene ontology analysis was performed to identify pathways that are
   enriched among genes that are differentially expressed at higher levels
   in PD and ILBD samples (padj < 0.05, RPKM in controls > 1, fold change
   > 1.5; [134]Figure 6 and [135]Supplementary Table 5). Terms related to
   negative regulation of apoptosis appears to be upregulated in PDearly
   samples. However, it should be noted that almost all genes contributing
   to these GO-terms belong to the same family of genes with homology to
   the mitochondrial encoded MT-RNR2 (humanin) gene ([136]Supplementary
   Figure 6). Humanin has been shown to have anti-apoptotic and
   neuroprotective effects and has been associated with Alzheimer’s
   disease ([137]Hashimoto et al., 2001). Several GO-terms relating to
   regulation of apoptosis and programmed cell death were also identified
   when analyzing enriched genes in PDlate ([138]Figure 6 and
   [139]Supplementary Table 5). Another set of GO terms among genes
   enriched in late PDlate samples were dominated by terms related to the
   response to unfolded proteins and chaperone-mediated protein folding.
   Genes associated with the endoplasmic reticulum (ER) unfolded protein
   response pathways (UPR) are included among these genes, e.g., XBP1,
   CHAC1, and DDIT3 (CHOP), but also many other genes encoding proteins
   that regulate cytoplasmic response to unfolded proteins
   ([140]Supplementary Figure 6 and [141]Supplementary Table 5). Enriched
   pathways identified from analysis of downregulated genes demonstrated
   that there was a significant downregulation of synaptic signaling and
   neuronal projection organization in PDlate samples suggesting defects
   in synaptic transmission and axon/dendrite integrity, which are
   hallmarks of PD pathology ([142]Supplementary Figure 7). Finally, in
   ILBD samples, downregulated genes were associated with GO terms
   relating to genes involved in cell junction. Strikingly, enriched genes
   were strongly linked to terms associated with oxidative phosphorylation
   ([143]Figure 6 and [144]Supplementary Table 5).

FIGURE 6.

   [145]FIGURE 6
   [146]Open in a new tab

   Up- and down-regulated biological processes in PDearly, PDlate and
   incidental Lewy body disease. Top significant biological process
   categories from differentially expressed genes (padj < 0.05, RPKM in
   controls > 1, fold change > 1.5) as analyzed by ToppGene suite. Heatmap
   indicates significance of each individual GO-term as revealed by
   Bonferroni test.

Discussion

   Understanding changes in gene expression in PD is important for several
   reasons. Identifying potentially dysregulated genes in PD can provide
   new biomarkers, information on pathological mechanisms, and potential
   novel therapeutic targets. Here we analyzed gene expression changes in
   post-mortem tissue samples from PD patients early after diagnoses; PD
   patients at advanced stages of disease; and ILBD patients,
   respectively. Our goal was to analyze gene expression specifically in
   mDA neurons of the SNc and for this reason LCM was used to isolate NM+
   cells from frozen tissue sections dissected from substantia nigra. The
   comprehensive analysis using RNAseq combined with LCM isolated cells
   has given novel insights into how gene expression changes during PD
   progression.

   Several previous studies have analyzed changes in gene expression in
   post-mortem brain tissue from PD patients ([147]Cantuti-Castelvetri et
   al., 2007; [148]Bossers et al., 2009; [149]Simunovic et al., 2009;
   [150]Zheng et al., 2010; [151]Elstner et al., 2011; [152]Riley et al.,
   2014). A relatively modest concordance is seen across different studies
   reflecting the challenges when analyzing differences in gene expression
   in patient samples ([153]Oerton and Bender, 2017). Several sources of
   variation are evident: (i) patient samples differ due to varying
   disease progression and clinical features of disease; (ii) differences
   in tissue quality, e.g., due to different PMIs is an additional source
   of variability; (iii) precision in dissection is challenging and
   unavoidably introduces substantial differences between studies; (iv)
   dissected tissue from substantia nigra is complex with regard to
   cellular composition and includes different neuronal and non-neuronal
   cell types. Of note, mDA neurons only comprise a minority of dissected
   cells and such cellular diversity further complicates interpretation of
   data, in particular when mRNA has been extracted from homogenized
   tissue samples consisting of mixed ventral midbrain cell types; (v) the
   method used for detecting gene expression varies between studies
   (hybridization arrays or RNAseq). Most importantly, different
   commercial hybridization platforms are well known to differ with regard
   to sensitivity and specificity in gene detection.

   Our goal has been to increase the precision in analysis by using LCM of
   NM+ mDA neurons to limit the variability in gene expression resulting
   from inclusion of surrounding non-DA neurons that may be more or less
   influenced by the disease process. In addition, quantifying gene
   expression by RNAseq has a superior sensitivity and dynamic range
   compared to hybridization arrays, which have been used in most previous
   studies. Moreover, the tissue samples used here are of high quality
   with relatively short PMI and have been annotated with regard to
   clinical features and disease progression ([154]Supplementary Figure
   1). Finally, our study included samples originating both from patients
   early after diagnosis and from more advanced stages of disease. The
   inclusion of tissue from early diagnosed PD patients allowed us to
   highlight the important question of how disease duration influences
   gene expression. Finally, the analysis of ILBD samples provided an
   interesting complement to PD and control samples.

   Our analysis showed that differentially expressed genes showed only a
   modest overlap when comparing changes in PDearly and PDlate samples,
   thus suggesting relatively clear differences in remaining NM+ mDA
   neurons dependent on disease duration. To understand the reason for
   these differences it is important to consider the rate of cell loss in
   PD. In a recent study, mDA neurons cell loss was studied in
   post-diagnostic intervals from 1 to 27 years ([155]Kordower et al.,
   2013). Importantly, a rapid (50–90%) loss of NM+ neurons was observed
   in the SNc in the first few years following diagnosis while only
   minimal additional loss is seen at later stages. Based on these
   histological data it can be concluded that the vast majority of
   remaining mDA neurons in PDlate samples are stably surviving NM+ mDA
   neurons since only minimal additional neuronal loss is seen at later
   stages ([156]Kordower et al., 2013). In contrast, at least a proportion
   of cells analyzed in PDearly samples are likely more acutely influenced
   by pathological stress as they will eventually degenerate. It seems
   likely that these differences are the basis for some of the substantial
   differences in gene expression observed in the two categories of PD
   samples. Accordingly, we hypothesized that most of the cells in PDlate
   samples are likely stably maintained in these PD patients and that gene
   expression in late PDlate samples, at least in part, can be explained
   by a progressive selection of cells that are more robust and with a
   higher propensity to survive over time in PD. Notably, in PDlate cases
   a majority of markers of human VTA mDA neurons were found to be
   enriched while a majority of the human SNc markers were downregulated
   ([157]Figure 5). This implies that mDA neurons with a subtype profile
   that is more similar to neurons of the VTA may represent a less
   vulnerable subpopulation that survives in SN of PD patients and for
   this reason contributes to the gene enrichment in samples isolated from
   late stage PD samples. Presumably, such VTA or “VTA-like” cells are
   normally localized in the more resilient dorsal tier of the SNc or in
   lateral parts of the VTA ([158]Fearnley and Lees, 1991). In contrast,
   it seems likely that differential gene expression in early samples, at
   least to some extent, reflects more immediate responses to cellular
   stress in the more vulnerable ventral tier of the SNc. Finally, it
   should be noted that the early post-diagnosis cases hade on average a
   relatively high age of disease onset (83 years) as compared to late
   post-diagnosis patients (68 years). This most likely reflects that
   death rarely occurs early after onset in younger patients and those
   samples are therefore uncommon in the tissue collection. However, we
   cannot rule out that these differences have also contributed to some of
   the differences in gene expression profiles seen between the two
   groups.

   In addition to the analyses of gene expression in mDA neuron samples
   isolated after short and long disease duration, respectively, our study
   also considered how gene expression in samples from individuals
   identified with ILBD related to PD and control samples. It remains
   unclear if ILBD represents a pre-clinical stage of PD or if it
   represents a unique pathological entity that does not cause PD symptoms
   ([159]Adler et al., 2010). It could be reasoned that a similar profile
   of up- or downregulated genes with those seen in PD < 4 years samples
   would be seen if ILBD represents a pre-clinical phase of PD. However,
   as ILBD has nigrostriatal dopaminergic losses midway between normal and
   PD, it seems a reasonable candidate for a preclinical stage and the
   differences between gene expression profiles seen in ILBD, PDearly and
   PDlate subjects may thus reflect distinct stages of one and the same
   disease ([160]Beach et al., 2008; [161]DelleDonne et al., 2008;
   [162]Dickson et al., 2008; [163]Iacono et al., 2015). If this is the
   case it is tempting to speculate that the striking upregulation of
   oxidative phosphorylation pathways seen in ILBD ([164]Figure 6) may
   indicate a neuroprotective response at early stages of disease
   development.

   Taken together, the results presented in this study provides a unique
   view of gene expression seen in remaining mDA neurons in patients with
   PD at different stages of disease duration in comparison to age-matched
   controls and ILBD cases. Further functional studies of identified
   regulated genes will be required to address their potential roles in
   relation to neuron vulnerability or neuroprotection.

Data Availability Statement

   The datasets generated and analyzed for this study can be found in the
   Gene Expression Omnibus, National Center for Biotechnology Information,
   using accession number [165]GSE182622
   .

Ethics Statement

   This study involving human participants was reviewed and approved by
   the Western Institutional Review Committee (WIRB), Seattle, Washington.
   The patients/participants provided their written informed consent to
   participate in this study.

Author Contributions

   TP and KT: conceptualization. KT, NV, LG, LD, and HL-M: formal analysis
   and investigation. TB, GS, and CA: resources. TP, KT, and NV: writing
   manuscript. KT and NV: computational analysis. TP: supervision. All
   authors contributed to the article and approved the submitted version.

Conflict of Interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

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Acknowledgments