Transcriptional evidence of reduced BDNF trophic signalling capacity in the post-mortem human midbrain of schizophrenia cases with high inflammation

doi:10.21203/rs.3.rs-4654926/v1

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Transcriptional evidence of reduced BDNF trophic signalling capacity in the post-mortem human midbrain of schizophrenia cases with high inflammation

https://doi.org/10.21203/rs.3.rs-4654926/v1

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Elevated inflammation in the midbrain of ~ 45% of people with schizophrenia may relate to altered trophic support for neurons in schizophrenia. Dopamine neurons require trophic support from Brain-Derived Neurotrophic Factor (BDNF), that signals via the full-length Tropomyosin kinase B receptor (TrkB^TK+). The truncated BDNF receptor (TrkB^TK−) may perpetuate neuroinflammation and the apoptosis-related p75 receptor may counteract the effects of BDNF. We hypothesised that transcriptional changes in either BDNF, and/or a transcription factor critical for the maintenance of dopamine neurons (Nuclear Receptor Related-1 protein; NURR1), and/or BDNF receptors – TrkB (TK + or TK-) and p75, would be found in the post-mortem schizophrenia midbrain, particularly in high inflammation cases. Using RT-qPCR, mRNA levels of NURR1, BDNF, TrkB and p75 were quantified from schizophrenia (n = 65) and control (n = 64) ventral mesencephalon. We found significant decreases in BDNF IV, TrkB^TK+ and NURR1 (14–18%) and increases in TrkB^TK− and p75 (18–35%) mRNA levels in schizophrenia compared to controls (all p < 0.05), with exacerbation in high inflammation schizophrenia. To determine whether these changes result from chronic antipsychotic treatment, we treated healthy adult rats with antipsychotics and found all corresponding trophic mRNAs to be unaltered. SnRNAseq of human midbrain showed that p75 receptor mRNA is primarily localised in oligodendrocytes and pan-TrkB mRNA is distributed to both neurons and astrocytes. We confirmed that p75 was localised to oligodendrocyte-like cells by immunohistochemistry. Altogether, we find transcriptional evidence of reduced levels of trophic support in the midbrain in schizophrenia and suggest that this may directly impact dopamine neuron health, particularly in neuroinflammatory conditions.

Biological sciences/Neuroscience/Molecular neuroscience

Health sciences/Diseases/Psychiatric disorders/Schizophrenia

BDNF

TrkB

schizophrenia

midbrain

inflammation

Excessive dopamine release by dopamine neurons projecting from the substantia nigra to the dorsal striatum is a robust neurobiological feature of schizophrenia, and putatively underlies psychotic symptoms [1, 2]. The higher energetic demands of increased dopamine release in schizophrenia may reduce the health, plasticity and viability of midbrain dopamine neurons. The excess dopamine may contribute to an increase in reactive oxygen species, which are known to disrupt cellular homeostasis [3] and induce neuroinflammation. Indeed, elevated pro-inflammatory markers in the midbrain of ~ 45% of people with schizophrenia is consistent with compromised dopamine neuron health [4, 5]. Dopamine neurons rely upon trophic factors such as Brain-Derived Neurotrophic Factor (BDNF) for support throughout development, maturation and survival [6–9]. BDNF is synthesised as preproBDNF, which is cleaved into proBDNF and mature BDNF [10]. Mature BDNF binds to the full-length Tropomyosin Kinase B receptor (TrkB^TK+) localised in neurons [11, 12] and to the truncated Tropomyosin Kinase B receptor (TrkB^TK−) localised mainly in glia [13, 14]. TrkB^TK+ activates pro-survival signalling cascades through phosphorylation of the intracellular tyrosine kinase domain and further upregulates BDNF expression [15, 16]. Co-expression of p75 with TrkB^TK+ in neurons promotes BDNF binding affinity [17]. While the p75 receptor can also bind to proBDNF to elicit apoptotic signalling [18, 19]the cellular localization of p75 in dopamine neurons in the human midbrain has not been established.

Significant reductions in BDNF and TrkB^TK+ (mRNA and protein) are present in multiple brain regions in schizophrenia, including the dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex and hippocampus [11, 20–26]. We hypothesised that these reductions in BDNF and TrkB^TK+ mRNA also exist in the ventral midbrain in schizophrenia, a region of concentrated dopamine neurons [27, 28]. BDNF expression is regulated by the transcription factor Nuclear Receptor Related 1 (NURR1), which induces the dopaminergic phenotype and maintains survival cues for dopamine neurons [29, 30]. We sought to determine whether NURR1 mRNA is reduced in schizophrenia, potentially providing further evidence that there may be less trophic signalling for dopamine neurons in the midbrain.

The interaction between neuroinflammation and trophic signalling in the midbrain in schizophrenia may also involve changes in the truncated isoform of the TrkB receptor (TrkB^TK−). This form of TrkB lacks the tyrosine kinase domain, and is predominantly localised in resident immune cells of the brain – astrocytes - and to a lesser extent, microglia [14, 31, 32]. TrkB^TK− expression on immune cells promotes gliosis and cytokine release, and diverts BDNF-mediated trophic support away from neurons [13, 33]. Additionally, TrkB^TK− can form dimers with TrkB^TK+ in neurons, where TrkB^TK− functions as a dominant-negative inhibitor to block pro-survival signalling [13, 34, 35]. TrkB^TK− mRNA is increased in the DLPFC in schizophrenia [36], and a similar percentage of people with schizophrenia display elevated pro-inflammatory markers (~ 40%) in this region [37]; however, it is unclear whether these increases in TrkB^TK− mRNA are concentrated in the subset of high inflammatory schizophrenia cases, or whether they occur more generally.

Hyperdopaminergia is evident in the midbrain in schizophrenia, and since inflammation may interact with BDNF to alter neuron plasticity and function, we hypothesised that BDNF and TrkB^TK+ mRNA would be reduced, whilst TrkB^TK− mRNA would be increased in the midbrain in schizophrenia, particularly in high inflammation conditions. We further hypothesised that the apoptotic p75 receptor and NURR1 transcription factor would be changed in the schizophrenia midbrain and these changes may relate to neuroinflammation. In order to determine the cellular source of BDNF, TrkB and p75, we interrogated our single-nucleus RNA sequencing (snRNAseq) results from the post-mortem human midbrain [38]. Additionally, the localisation of the p75 receptor protein was established using immunohistochemistry to determine whether this receptor could directly affect midbrain dopamine neurons. To test whether changes in neurotrophins may result from chronic antipsychotic treatment, we chronically administered typical and atypical antipsychotics to healthy adult male rats and then assessed gene expression of trophic signalling molecules in the substantia nigra.

Post-mortem human midbrain tissue collection and demographics

All experimental protocols using human brain tissue were approved by the University of New South Wales (UNSW) Human Research Ethics Committee (HREC#12435). For homogenate-based work, post-mortem midbrain tissue was provided by the NSW Brain Tissue Resource Centre (TRC, Sydney, Australia) (n = 30 schizophrenia and n = 30 control cases) and Stanley Medical Research Institute (SMRI, Maryland, USA) (n = 35 schizophrenia and n = 35 control cases) (Table 1). Ventral midbrain tissue was dissected from six 60µm cryostat sections cut in the coronal plane at the level of the oculomotor nerve exit. Cases for gene expression analysis were previously characterised as being in either a low or high immune cluster based on two-step recursive cluster analysis of SERPINA3, IL6, IL1β and TNFα mRNAs in the midbrain [4, 5]. A randomly selected subgroup of cases from the SMRI cohort was used for snRNAseq to determine the cellular distribution of BDNF, TrkB and p75 mRNA expression (14 control, 20 schizophrenia cases) (Table S1). The TRC cohort was used for immunohistochemical analysis of p75 receptor protein expression in the midbrain (Table S2).

Diagnosis for schizophrenia was made according to DSM-IV and cases were matched with controls on the basis of age, sex, RNA Integrity Number (RIN), and post-mortem interval (PMI) within cohorts and did not differ in the combined cohort, whereas brain pH was significantly lower in cases compared to controls (Table 1). All but 2 schizophrenia patients were known to have received antipsychotic treatment. Where available, antipsychotic measurements (mean daily, lifetime and last dose) were converted to chlorpromazine (CPZ) equivalents and are recorded with duration of illness in schizophrenia patients (Table 1, Table S1-2). In the combined SMRI and TRC cohort, 45 schizophrenia patients were treated with typical antipsychotics, 18 were treated with atypical antipsychotics, and the clinical medication history of 2 cases were unknown.

Animal treatment and tissue extraction

All experimental protocols on animals were approved by the UNSW Animal Research Ethics Committee (#21/26A). Adult male Sprague Dawley rats (Animal Resource Centre, WA, Australia) were habituated from P60 to twice daily treatment with drug-free cookie dough for 1 week prior to being assigned treatment to either cookie dough with 2mg/kg/day haloperidol, or 2mg/kg/day risperidone added, or control cookie dough without antipsychotic (n = 14/group). Rats were weighed weekly and cookies were made to respective weights ± 10%. Haloperidol (Serenace 5mg tablets, Aspen Pharma Australia) and Risperidone (Rixadone 4mg tablets, Alphapharm) were crushed for supplementation in cookie dough [35% cornflour, 10% gelatine powder, 35% icing sugar, 20% milk powder dissolved in MilliQ water (17% mL/g)] [39–41] and administered twice daily for 7 months to resemble the extended antipsychotic exposure that humans with schizophrenia would receive relative to their corresponding life-span (mean ~ 20 years duration of treatment in humans [42]). Following the treatment period, animals were anaesthetised with isoflurane in a gas chamber prior to decapitation. Brains were excised, sectioned coronally, snap frozen in isopentane (Sigma-Aldrich) and stored at -80°C. To extract midbrain tissue, the midbrain block was faced on the cryostat until the anterior landmark for the substantia nigra was visualised around bregma − 4.56mm [43]. Ventral midbrain tissue was dissected from five 100µM thick sections for RNA extraction.

RNA extraction and quantitative real-time polymerase chain reaction (qPCR) analysis

Midbrain tissue from human (19–83 mg) and rodent samples (~ 20mg) was homogenised in TRIzol (Life Technologies, Scoresby, VIC, Australia). Quantity and quality of total RNA was determined using Nanodrop ND 1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). RIN was measured with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Superscript IV First Strand Synthesis Kit (Life Technologies) and random hexamers were used to synthesise cDNA, following the manufacturers’ instructions. TaqMan gene expression assays (Applied Biosystems, Life Technologies, Foster City, CA, USA) were used for qPCR analysis of BDNF IV, NURR1, TrkB^TK+, TrkB^TK- and p75 in the human (Table S3) and rodent cohort (Table S4). BDNF exon IV was assessed due to its relatively higher expression level in the substantia nigra compared to other exons and activity-dependent transcriptional profile [44, 45]. Human gene expression was measured in a 1:5 dilution of cDNA using high-throughput integrated fluidic circuits by Fluidigm Biomark HD (Fluidigm Corporation, CA, USA) at the Ramaciotti Centre for Genomics (UNSW, Sydney, Australia). A dilution series of cDNA pooled from all samples was included to determine gene expression in samples using the relative standard curve method (n = 8 points), as previously described [46]. Controls without template and reverse transcriptase were included on each plate. Fluidigm Real-Time PCR Analysis software version 4.5.2 (Fluidigm Corporation, CA, USA) was used to extract and analyse qPCR data, with the quality threshold set at 0.65. Genes of interest were normalised to the geomean of 4 housekeepers: beta-glucuronidase (GUSB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), TATA-box binding protein (TBP) and ubiquitin C (UBC), which were unchanged between diagnostic groups in the human cohort (p > 0.05; Fig. S1). For the rodent cohort, each sample was assayed in triplicate using the ABI Prism 7900HT System (Applied Biosystems, Life Technologies). Serial dilutions of cDNA pooled from all samples were included as 8 standards and the relative standard curve method was used to quantify gene expression using Sequence Detector Software (V2.4, Applied Biosystems). Genes of interest were normalised to the geomean of GAPDH, GUSB, β-actin (ACTB) and tyrosine-3-monooxygenase (YWHAZ) and were unchanged by treatment (p > 0.05; Fig. S2).

SnRNAseq

Ventral midbrain tissue was extracted from five 100µm sections of a sub-cohort of cases from SMRI. Tissue was lysed in a sucrose lysis buffer, filtered, and nuclei were purified by ultracentrifugation in a dense sucrose buffer, as previously described [38, 47]. Tissue quality was assessed from adjacent sections to sampled tissue using RNeasy Lipid Tissue mini kit (Qiagen, 75804). Fluorescence activated sorting of nuclei identified DAPI-positive nuclei, which were negative for the early transcription marker for oligodendrocyte differentiation Olig2 (Anti-OLIG2 clone 211F1.1 AF488 mouse mAB, Merck Millipore, MABN50A4) and were also NeuN depleted, with only ~ 1 out 6 DAPI-positive nuclei also positive for the neuronal marker NeuN (1B7 AF647 mouse mAB, NBP1-92693AF647, Novus Biologicals). Single nucleus cDNA libraries were constructed with Chromium Single Cell 3’ Reagents Kit v3 (10x Genomics), according to the user guide. Equimolar ratios of pooled samples were sequenced on a NextSeq 500 platform (Research Sequencing Facility, University Medical Centre, Groningen, Netherlands). Sequencing reads were processed and aligned to the GRCh38 human genome (release 110 downloaded from Ensembl ftp) using CellRanger 7.0.1, with both exonic and intronic reads included, along with basic quality control analyses using fastqc program [48]. The mean sequencing depth was 223 million reads per sample, and the mean reads per nuclei was 43,263. The mean estimated number of nuclei was 5,184 per sample and the mean total detected genes was 38,326. Count matrices filtered in CellRanger were input to R v4.3.2 with Seurat v5.0 [49]. Nuclei with > 5% mitochondrial content were removed. Count information was log normalized and integrated with reciprocal principal component analysis using Seurat. Scrublet v0.2.1 was used to remove doublets with default settings. Unbiased clustering analysis was performed using Find Neighbors (dim = 1:30, k.param = 10) FindClusters function with resolution set to 0.4 and major cell types were manually defined based on the expression of marker genes (Table S5). Expression levels of specific genes were visualized using feature plots in Seurat.

Immunohistochemistry

Fresh frozen midbrain sections from all TRC cases were used to visualise p75 receptor protein expression. Slices (14µM) were cut using a cryostat (Leica 3050M) at the level of the oculomotor nerve, collected onto gelatine-subbed slides, stored at -80^oC and then thawed at RT for 20 min. Tissue was fixed in 4% paraformaldehyde in PBS, rinsed in PBS, then 30% MeOH with 3% H₂O₂ for 20 min to block endogenous peroxidase activity. Following additional PBS washes, slides were blocked with 10% normal horse serum (S2000, Vector Laboratories, CA, USA) diluted in PBS, 0.05% bovine serum albumin and 10% Triton for 1 hr. Primary antibody for p75 (1:800, rabbit anti-p75 ab52987, Abcam, Melbourne, VIC, AUS) was applied overnight at 4 ^oC. Slides were rinsed in PBS and incubated with a 1:500 dilution of horse anti-rabbit IgG-biotinylated secondary antibody (BA200, Vector Laboratories) for 1 hr at RT. After rinsing and incubating with the avidin-biotin-peroxidase complex (PK4000, Vector Laboratories) for 1 hr at RT, 3,3’-diaminobenzidine solution (DAB D5637, Sigma-Aldrich, MO, USA; final concentration 12 mmol/L in PBS with 0.003% H₂O₂;) was applied for 2 min. A final PBS wash was followed by dehydration with an ethanol gradient and Nissl counterstaining (0.02% thionine), prior to coverslipping. Slides were visualised under a Nikon Eclipse 80i light microscope using the 20X objective and brightfield settings.

Statistical analysis

Statistical tests were performed using SPSS (V26, IBM, Armonk, NY, USA), with the threshold for statistical significance set to p < 0.05. Outliers greater than two standard deviations from the diagnostic and immune subgroup mean in the human cohort and from treatment group mean in the rodent cohort were excluded. Shapiro-Wilk and Levene’s test assessed for normality and homogeneity of variance of mRNA levels respectively within diagnostic and immune subgroups in human cohorts and within treatment groups in the rodent cohort. If data did not satisfy normality in any group, data was log₁₀ transformed and re-tested for normality. Pearson’s and Spearman’s correlations were used to test any association between demographic variables with mRNA levels in human cohorts. As altered pH is implicated in the schizophrenia disease state, pH was not used as a covariate [50]. Where clinical history was available, Spearman’s correlations between gene expression with lifetime, daily and last CPZ-equivalent antipsychotic dose as well as duration of illness were calculated for schizophrenia cases. ANCOVA and Quade’s rank analysis was used to analyse normally and non-normally distributed gene expression data respectively with covariates. Where there were no covariates, Independent Samples t-test was used to test for diagnostic differences and ANOVA to test for immune subgroup differences, with Fisher’s Least Significant Difference post-hoc test, to assess gene expression in the human cohort. Where normality was not achieved, non-parametric Mann-Whitney U was used for comparison of mRNA levels by diagnosis or Kruskal-Wallis tests were used to compare mRNA levels between immune subgroups of the human cohort or by treatment group in the rodent cohort. Graphs were generated using GraphPad Prism 9 (GraphPad Software, La Jolla, CA).

Gene expression correlations with demographic and clinical variables in the post-mortem human midbrain

BDNF IV, TrkB^TK+ and NURR1 mRNAs positively correlated with RIN across the combined post-mortem cohorts used for qPCR (all r > 0.20; p < 0.05) (Table S6). TrkB^TK+ (r = 0.209) and p75 (r= -0.235) mRNAs also negatively correlated with PMI (p < 0.05) (Table S6).. As such, RIN was used as a covariate for BDNF IV, TrkB^TK+ and NURR1 gene expression analysis between diagnostic and immune subgroups, and PMI was used as a covariate for TrkB^TK+ and p75 mRNA analysis by diagnosis and immune subgroup. In the schizophrenia group, gene expression of BDNF IV (r= -0.347, p = 0.01) and NURR1 (r= -0.269, p = 0.036) negatively correlated with duration of illness and p75 mRNA positively correlated with daily CPZ-equivalent antipsychotic dose (r = 0.431, p = 0.045) (Table S7).

Reductions in BDNF IV, NURR1 and TrkB ^TK+ and increases in TrkB^TK- and p75 mRNAs in the midbrain in schizophrenia are magnified in high inflammation cases

Gene expression of BDNF IV and its upstream transcription factor, NURR1 was reduced by 18% [F_(1,110) = 7.591, p = 0.007] and 14.9% [F(_1,115) = 6.867, p = 0.01] respectively in the schizophrenia group compared to controls (Fig. 1a-b). Gene expression of the primary full-length neurotrophic receptor of BDNF, TrkB^TK+, was also reduced in schizophrenia cases compared to controls by 18.5% [F(_1,116) = 19.768, p < 0.001] (Fig. 1c). In contrast, mRNA levels of the truncated TrkB^TK− isoform were increased by 18.6% in schizophrenia cases compared to controls [F(_1,117) = 7.499, p = 0.007] (Fig. 1d). Apoptotic p75 receptor mRNA levels were also increased by 34.7% [F(_1,114) = 19.89, p < 0.001] in schizophrenia cases compared to controls (Fig. 1e), reflecting the greatest magnitude of change evident across diagnostic groups of the receptors assessed.

BDNF IV [F(_2,110) = 3.893, p = 0.023], NURR1 [F(_2,111) = 6.283, p = 0.003], TrkB^TK+ [F(_2,113) = 15.07, p < 0.001], TrkB^TK− [F(_2,113) = 5.959, p = 0.003] and p75 [F(_2,112) = 11.624, p < 0.001] mRNA levels were also significantly changed according to inflammatory subgroups (Fig. 2). More specifically, there was a greater mean decrease in BDNF IV (30.8%; p = 0.007), NURR1 (30.1%; p = 0.001) and TrkB^TK+ (23.5%; p < 0.001) mRNA levels when comparing high inflammation schizophrenia cases to control cases than when just examining diagnostic changes alone and TrkB^TK+ mRNA was also decreased in low inflammation schizophrenia cases compared to controls (p = 0.011). However, the levels of BDNF and NURR1 mRNAs in the low inflammation group compared to controls did not achieve statistical significance, despite having a similar number of subjects. A similar pattern of magnified change was observed for transcripts that were increased in the schizophrenia midbrain, where those with a high inflammatory status had a 46.3% increase in p75 levels (p < 0.001) and 38% increase in TrkB^TK− levels (p = 0.001) mRNA compared to controls. TrkB^TK− mRNA was also increased in the high inflammation schizophrenia subgroup compared to the low inflammation schizophrenia subgroup (p = 0.019). P75 mRNA levels were also significantly increased in the low inflammation schizophrenia subgroup compared to controls (p = 0.001). Consistent with actions of the pro-trophic ligand, receptor, and transcription factor, BDNF mRNA levels positively correlated with TrkB^TK+ (r = 0.563) and NURR1 (r = 0.419) mRNA levels (p < 0.0001) (Table S8). In contrast, BDNF mRNA levels negatively correlated with TrkB^TK− (r= -0.307) and p75 receptor (r= -0.362) mRNA levels (p < 0.001). P75 mRNA negatively correlated with NURR1 (r= -0.142, p = 0.014) and TrkB^TK+ (r= -0.362, p < 0.0001) mRNA, yet was not correlated with TrkB^TK− mRNA (r= -0.097, p = 0.293).

Cell-type specific expression of BDNF, TrkB and p75 receptors

SnRNAseq data of nuclei isolated from controls and schizophrenia cases were analysed together for unbiased cluster analysis of transcriptomic profiles from ~ 124,416 nuclei and yielded 33 clusters of cell types within the ventral midbrain (Fig. 3a). Top 10 transcripts used to characterise each cluster of cells are detailed in Table S5. Nuclei with similar transcriptomic profiles were grouped together, with some clusters containing multiple cell types. Neurons were the most prevalent cell type, spread across multiple clusters, followed by astrocytes, microglia/macrophages and oligodendrocytes (low due to excluding Oligo2 + nuclei). Unbiased cluster analysis of the transcriptome of nuclei within control, low and high inflammation schizophrenia subgroups revealed a similar profile of clusters across all subgroups (Fig. 3b-d). Comparison of BDNF gene expression within clusters across immune subgroups of schizophrenia cases and controls revealed that BDNF mRNA was primarily localised to clusters containing neurons across all three subgroups, with occasional expression of BDNF evident in some astrocyte subtypes (Fig. 3b). High levels of BDNF mRNA were also observed in some microglia/macrophages in high inflammation schizophrenia cases, but not in low inflammation schizophrenia or control cases. Pan-TrkB mRNA, comprised of both TrkB^TK+ and TrkB^TK- isoforms, was ubiquitously expressed across multiple cell types, with similar levels of expression in neurons and astrocytes across all three immune subgroups. Pan-TrkB expression was also evident in clusters containing fibroblasts, oligodendrocytes, endothelial cells, T cells and to a lesser extent microglia/macrophages and pericytes (Fig. 3c). In contrast, gene expression of the p75 receptor was limited to the cluster containing oligodendrocytes (Fig. 3d). Levels of p75 receptor expression was sparse and was increased in several subtypes of oligodendrocytes in low and high inflammation schizophrenia subgroups compared to controls (Fig. 3d)

P75 receptor protein is primarily localised to oligodendrocytes in the post-mortem midbrain

Using immunohistochemistry, p75 protein was anatomically localised to the substantia nigra of all individuals in the TRC cohort (Fig. 4a). P75 receptor immunoreactivity was not evident in diffuse Nissl-stained nuclei of neuromelanin-containing dopamine neurons of the midbrain in either control or schizophrenia cases, nor was it found in neuron-like cells devoid of neuromelanin. However, in both schizophrenia and control cases, the clearest signal for p75 protein in the midbrain was detected in putative oligodendrocytes, which were histologically identified through a round cell body and lighter Nissl-stained nuclei compared to other glia-like cells, which are smaller and more intensely Nissl-stained [51, 52]. Oligodendrocytes positive for p75 immunostaining were most prominent in white matter of the midbrain sections (Fig. 4b). Dark p75 receptor immunoreactivity was evident in white matter tracts of the cerebral peduncles in 18% of controls (5/27 cases), 33% of low inflammation (5/15 cases) and 50% of high inflammation (6/12 cases) schizophrenia cases (Fig. 4c)

Chronic antipsychotic treatment does not alter BDNF IV, NURR1, TrkB ^TK+, TrkB^TK− or p75 mRNA in the midbrain of healthy adult rats

Gene expression levels of BDNF IV [H(₂) = 0.881, p = 0.644] and its transcription factor NURR1 [H(₂) = 1.663, p = 0.435] were unaltered in midbrain tissue homogenates obtained from normal adult rats chronically treated with typical or atypical antipsychotics for 7 months (Fig. 5a-b). However, the haloperidol treatment group had a non-significant mean increase in BDNF IV mRNA (41.19%) and NURR1 mRNA (46.46%) compared to controls, which is the opposite direction of change found in schizophrenia cases in the human post-mortem substantia nigra. Chronic antipsychotic treatment did not statistically change mRNA levels of BDNF receptors in the ventral midbrain, including TrkB^TK+ [H(₂) = 3.224, p = 0.199], TrkB^TK− [H(₂) = 0.116, p = 0.944] and p75 [H(₂) = 0.005, p = 0.998] (Fig. 5c-e).

This study is the first to identify transcriptional changes reflective of reduced trophic support in the schizophrenia midbrain, and these changes are more robust in the presence of neuroinflammation. We find that reductions in trophic support proximal to midbrain dopamine neurons in schizophrenia include reduced BDNF and TrkB^TK+ gene expression. We predict that these would also be present at the protein level, as is found in other brain regions in schizophrenia [23, 36]. Our finding of reduced NURR1 mRNA, especially in high inflammation schizophrenia cases, supports our hypothesis that these altered gene expression levels could impact the health and maintenance of dopamine neurons. In contrast, we find increases in “negative regulators” for both TrkB^TK− and p75 receptor mRNA. While these mRNAs are likely to be localised to astrocytes and oligodendrocytes respectively, and therefore may not directly impact BDNF signalling on midbrain dopamine neuronal cell membranes. We posit that these putative changes in mRNA may impede neurotrophic signalling and could impact neurons, including dopamine neurons, of the substantia nigra.

Overall downregulation of BDNF mRNA in the schizophrenia group compared to controls in this study appears to be mostly found in cases in the high inflammatory subgroup, as corresponding reductions in BDNF mRNA are not present in schizophrenia cases with low inflammation. Pro-inflammatory cytokines compromise the capacity of BDNF to provide trophic support to neurons by interfering with signal transduction from the TrkB^TK+ receptor to PI3K/Akt and Ras/ERK pathways, which are responsible for the neuroprotective effects of BDNF [53–55]. SnRNAseq confirmed a primarily neuronal profile of BDNF expression in the post-mortem human midbrain in both low and high inflammation schizophrenia cases, as well as controls. However, further investigation into the specific subtypes of neurons where reductions in BDNF mRNA are localised in schizophrenia midbrain is warranted.

Dysregulated transcription of BDNF in schizophrenia may be directly related to NURR1 as NURR1 is a transcription factor for BDNF and NURR1 mRNA is also decreased in the midbrain in schizophrenia cases with high inflammation. NURR1 is critical for dopamine neuron survival as NURR1 deficiency and rare variants of the NURR1 gene contribute to neurodegeneration of dopamine neurons in Parkinson’s disease [56, 57]. Decreased NURR1 expression can precede downregulation of BDNF mRNA and is paralleled by reductions in TH in cultured mesencephalic neurons [77], which are less resilient to neurotoxicity with NURR1 deficiency [58], reflecting specific impediments to dopamine neuron survival mechanisms [59]. Hence, the observed reductions in NURR1 mRNA in the midbrain of high inflammation schizophrenia cases supports another feature of impaired trophic support for midbrain dopamine neurons and suggests compromised health of dopamine neurons in schizophrenia.

Further evidence for disrupted BDNF-mediated trophic signalling in the midbrain are reductions in TrkB^TK+ mRNA in both of the inflammatory schizophrenia subgroups compared to controls, demonstrating that reduced TrkB^TK+ mRNA may be detected in the absence of overt tissue inflammation in schizophrenia. Using snRNAseq, we detected pan-TrkB (both TK + and TK- splice variants) across multiple subtypes of neurons and astrocytes. This is consistent with the TrkB^TK− splice variant being principally localised to glial cells in the human DLPFC [32] and also supports that alterations in full-length TrkB mRNA can affect both dopaminergic and non-dopaminergic neurons in the midbrain. Indeed, BDNF and TrkB reductions are correlated with deficits in inhibitory neuron transcripts in the cortex of people with schizophrenia [21]. We have found evidence of GABA neuron dysfunction, marked by reductions in glutamate decarboxylase mRNA and protein, as well as reduced parvalbumin mRNA, in schizophrenia midbrain [60], suggesting these deficits could also be correlated with neurotrophin reductions in subcortical regions. This reduction in TrkB^TK+ mRNA could also impact dopamine neurons as TrkB hypomorphic mice with approximately 25–30% of normal levels of TrkB receptors display dopamine neuron loss in the nigrostriatal pathway [61], with accompanying gliosis [62]. We find levels of TrkB^TK+ mRNA in schizophrenia are not as profoundly changed as in hypomorphic mice (~ 75% that of controls), yet this deficit could have a detrimental impact on midbrain dopamine neurons; however, a more extensive analysis of dopamine neurons in schizophrenia is needed.

In contrast to the full-length TrkB receptor, mRNA levels of the truncated receptor, TrkB^TK−, were elevated in the midbrain of schizophrenia cases compared to controls, primarily in cases with high inflammation. BDNF binding to TrkB^TK− receptors on glia can promote the release of pro-inflammatory cytokines, including IL1β and TNFα, consistent with the increase of these cytokines at the mRNA and protein level we have found in the schizophrenia midbrain [54, 63–65]. We also find increased reactive astrocytes (GFAP) and microglia (IBA1) mRNAs in schizophrenia midbrain, and these cells are proposed mediators of the neuroinflammatory state in schizophrenia [5, 66]. Inflammatory activity of resident immune cells of the brain can also regulate neurotransmission as cytokines can evoke dopamine release and further perpetuate the hyperdopaminergic state of nigrostriatal dopamine neurons, and thereby may contribute to psychotic symptoms in schizophrenia [67, 68].

Interestingly, increases in p75 receptor mRNA in schizophrenia midbrain appear to transcend inflammatory state and are found in both high and low immune subgroups. This was an unexpected finding since p75 is upregulated in neurons and astrocytes in response to inflammation and oxidative stress in vitro [69, 70]. Also, in contrast to our expectations, snRNAseq of midbrain nuclei demonstrated that p75 receptor gene expression appears to be found mostly in oligodendrocytes in schizophrenia cases and controls. This observation was confirmed with immunohistochemistry; we found p75 receptor protein in oligodendrocyte-like cells and white matter tracts in many of the low and high inflammation schizophrenia cases, as well as some control cases. As p75 receptor expression in oligodendrocytes has a dual role, mediating both regenerative potential of these cells, but also promoting cell death [71, 72], these changes are difficult to interpret. While it is possible that increased p75 may be beneficial to oligodendrocytes in schizophrenia midbrain in the absence of neuroinflammation, we speculate that oligodendrocytes may be vulnerable to cell damage/death in high inflammation schizophrenia as mRNA for the cell-death ligand, TNFα, which is known to bind to p75, is increased. Indeed, myelin dysfunction, together with reductions in subcortical and cortical oligodendrocytes and associated transcripts, is found in schizophrenia in association with neuroinflammation [73–78]. Altogether, p75 receptor upregulation in the schizophrenia midbrain appears primarily localised in oligodendrocytes, indicating a role for the p75 receptor in contributing to white matter pathology in schizophrenia, rather than directly interfering with BDNF-mediated trophic support on the cell membrane of midbrain neurons.

We have evidence that the transcriptional changes evident in midbrain BDNF, TrkB, p75 and NURR1 may not result from long-term antipsychotic exposure as we do not find any changes in these mRNAs in healthy adult rats chronically treated with either first- (haloperidol) or second-generation (risperidone) antipsychotic drugs. However, animal studies assessing the effects of chronic antipsychotic exposure with a shorter duration of treatment report reductions in BDNF in multiple brain regions [79–83]. In humans, we find that midbrain p75 mRNA positively correlated with daily antipsychotic dose, supporting a potential contribution of antipsychotics to p75 receptor upregulation. However, midbrain BDNF mRNA did not correlate with antipsychotic exposure, consistent with previous post-mortem studies which also report no significant correlations with lifetime antipsychotic doses [23, 24, 26]. Reductions in TrkB^TK+ mRNA levels in the post-mortem human DLPFC in schizophrenia are found and correlate with higher lifetime antipsychotic dose [11], so the potential impact of antipsychotics cannot be entirely ruled out. Gene expression of BDNF and NURR1 mRNA in the midbrain was found to negatively correlate with duration of illness in schizophrenia cases, and this may reflect that both mRNAs are known to decrease with age [84, 85], and the effect of increasing age on the brain may be more accelerated in schizophrenia [86, 87]

Limitations

Nuclei selected for snRNAseq analysis were Olig2- and NeuN+, suggesting that many cells that may express BDNF, TrkB and p75 were not selected for analysis, including subpopulations of dopaminergic neurons that are NeuN- in mammals [88] and oligodendrocytes. Future snRNAseq analysis may instead select for cells that express a marker for dopamine neurons to specifically characterise the transcriptional landscape in midbrain dopamine neurons in schizophrenia. Additionally, this study assessed the effects of chronic antipsychotic treatment on midbrain trophic transcripts in healthy adult rats; however, antipsychotics may differentially regulate trophic support molecules in the schizophrenia disease state, particularly when neuroinflammation is present.

Here, we identified transcriptional reductions in beneficial neurotrophic factors in the schizophrenia midbrain, many of which are exacerbated by neuroinflammation and do not appear to be a direct consequence of chronic antipsychotic treatment. Increases in the truncated TrkB^TK− receptor may also compromise neurotrophic function in schizophrenia midbrain. Increased p75 receptor activity, likely in oligodendrocytes, in schizophrenia supports a role for the p75 receptor in these cells rather than altering trophic signalling within midbrain neurons. Further studies aiming to explore other hallmarks of dopamine neuron health, particularly in the presence of neuroinflammation, will inform therapeutic strategies to preserve cellular integrity and ameliorate potential functional deficits ensuing from the prospective loss of neurotrophic support in schizophrenia.

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Transcriptional evidence of reduced BDNF trophic signalling capacity in the post-mortem human midbrain of schizophrenia cases with high inflammation

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Abstract

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Introduction

Methods

Post-mortem human midbrain tissue collection and demographics

Animal treatment and tissue extraction

RNA extraction and quantitative real-time polymerase chain reaction (qPCR) analysis

SnRNAseq

Immunohistochemistry

Statistical analysis

Results

Gene expression correlations with demographic and clinical variables in the post-mortem human midbrain

Cell-type specific expression of BDNF, TrkB and p75 receptors

P75 receptor protein is primarily localised to oligodendrocytes in the post-mortem midbrain

Discussion

Limitations

Conclusion

References

Table 1

Additional Declarations

Supplementary Files

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