Dopaminergic Changes in the Subgenual Cingulate Cortex in Dementia with Lewy Bodies Associates with Presence of Depression

Dopaminergic Changes in the Subgenual Cingulate Cortex in Dementia with Lewy Bodies Associates with Presence of Depression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Dopaminergic Changes in the Subgenual Cingulate Cortex in Dementia with Lewy Bodies Associates with Presence of Depression Lina Gliaudelytė, Steven Rushton, Alan Thomas, Rolando Berlinguer Palmini, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3953937/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In addition to the core clinical features of fluctuating cognition, visual hallucinations, and parkinsonism, individuals with dementia with Lewy bodies (DLB) frequently experience chronic and debilitating major depression. Treatment of depression in DLB is hampered by a lack of available effective therapies and standard serotonergic medication for major depressive disorder (MDD) is typically ineffective. Dysfunction of dopaminergic neurotransmission contributing to anhedonia and loss of motivation has been described in MDD. The subgenual anterior cingulate cortex (sgACC) is important in mood regulation and in the symptomatic expression of depression, displaying structural, functional and metabolic abnormalities in MDD. To assess dopaminergic and serotonergic synaptic changes in DLB, post mortem sgACC tissue from DLB donors with and without depression was investigated using high-resolution stimulated emission depletion (STED) microscopy, as well as Western and dot blotting techniques. STED imaging demonstrated the presence of α-synuclein within individual dopaminergic terminals in the sgACC, α-synuclein presence showing a significant positive correlation with increased SNAP25 volumes in depressed DLB cases. A reduction in dopaminergic innervation in the sgACC was observed in DLB cases with depression, along with reduced levels of multiple dopaminergic markers and receptors. Limited alterations were observed in serotonergic markers. Our work demonstrates a role for dopaminergic neurotransmission in the aetiology of depression in DLB. Careful and selective targeting of dopaminergic systems may be a therapeutic option for treatment of depression in DLB. Biological sciences/Neuroscience/Molecular neuroscience Health sciences/Biomarkers/Predictive markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Dementia with Lewy bodies (DLB) is a significant cause of morbidity in older populations, representing between 5–20% of all clinical dementia cases 1, 2 . DLB shows core symptoms of fluctuating cognition, parkinsonism, the presence of recurrent complex visual hallucinations and REM sleep behaviour disorder 3 . Psychiatric features are highly prevalent in many DLB patients, with major depression present in 50–80% of cases 4 . Depression is frequently present at prodromal DLB stages, persisting throughout the clinical course 5 . Depression in dementia is associated with poor quality of life, increased morbidity and more rapid cognitive decline, and treatment would have significant patient benefit 6, 7 . The subgenual anterior cingulate cortex (sgACC) is a key brain region associated with mood and anxiety, and relays emotional information between limbic, cortical and subcortical regions 8, 9 . Abnormalities in sgACC activity, connectivity and grey matter volume have been shown in depressive disorders 10 , with sgACC used as a target for treating treatment resistant depression with deep brain stimulation 11, 12 . The monoamine hypothesis of depression in unipolar major depressive disorder (MDD), suggests that reduction in serotonergic and noradrenergic neurotransmission underpins depressive symptoms 13 . Considerable evidence supports the monoamine hypothesis in individuals with MDD, with reduced 5HT concentrations observed in serum and reduced 5HIAA in CSF in patients, along with decreased serotonin 5HT1 A and 5HT2 A binding in anterior cingulate cortex (ACC) in MDD 14, 15 . However, reduced levels of 5HT as a basis of depression have been challenged by some, primarily based on reduced and delayed response to 5HT based therapies 16, 17 . Data on serotonergic treatment of depression in DLB is lacking, and in depression in Parkinson’s disease (PD) serotonergic treatment shows minimal effects in a few small-scale trials 18, 19 . Dopamine plays a role in reward and stress 20 with dysfunction of dopaminergic neurotransmission within the mesolimbic and mesocortical systems contributing to anhedonia and loss of motivation in depression 21 . A significant reduction in dopamine transporter (DAT) uptake is observed in anhedonic depressed patients 22 , along with increased striatal D2 receptor binding in MDD suggesting decreased dopamine turnover 23 . Depression in PD is associated with nigral and mesolimbic dopaminergic pathway dysfunction with reduced ventral tegmental area (VTA), cingulate and amygdala volumes on MRI, and reduced [11C]RTI-32 DAT uptake in limbic regions in PD patients, with depression correlated with loss of dopamine projections from the VTA 24 and substantia nigra (SN) 25 . Whilst PD with dementia shows reduced VTA neurone numbers, altered VTA neurone number in DLB is mild although neurone dysfunction has not been established (Patterson et al 2018). However, both DLB and PD show SN dopaminergic neurone loss and the contribution of SN dopamine depletion is unknown (Patterson et al 2018). What role dopaminergic systems plays in DLB patients with depression is unclear. In this study, we investigated dopaminergic parameters in relation to depression in DLB. We focussed on the subgenual anterior cingulate cortex (sgACC) due to its important role in mood regulation and the symptomatic expression of depression, and as a region vulnerable to α-synuclein pathology in DLB 10, 26 . Little is known about how changes in dopaminergic neurotransmission in the sgACC might relate to development of depression in DLB, despite high levels of α-synuclein and other neurodegenerative pathology within the sgACC 27 . We therefore assessed dopaminergic innervation in the sgACC using a combination of stereology, high resolution stimulated emission depletion (STED) microscopy, and protein determination using post-mortem tissue samples from DLB cases with and without depression to investigate associations with depression. Materials and Methods Clinical Information Post-mortem tissue was obtained from the Newcastle Brain Tissue Resource (NBTR). Ethical approval was granted by Newcastle and North Tyneside-1 National Health Service (NHS) Research Ethics Committee. Donors had received clinical assessments during life and consented to the use of brain tissue for research purposes. Seventeen controls, 15 DLB cases without depression and 13 DLB cases with depression were included, with 12 from each group used for biochemical analysis. Inclusion criteria for depression diagnosis used the Cornell Scale for Depression in Dementia (CSDD) (score ≥ 8) as a validated rating scale. Alternatively, the Geriatric Depression Scale (GDS) (score ≥ 10) was used if CSDD scores were not available (see Supplementary Methods 1). Immunohistochemistry Formalin fixed paraffin embedded tissue blocks from the right hemisphere containing the sgACC (Brodmann area: BA25) sampled at the rostrum of the corpus callosum were cut at 10µm using a rotary microtome. Immunohistochemical staining used established protocols (see Supplementary Methods 2). For STED microscopy, sections were blocked using 10% goat serum (NGS: Sigma G9023) for one hour at room temperature, followed by incubation with primary antibodies overnight at 4°C. After washes in TBS, sections were incubated with secondary fluorescent antibodies in TBS and 10% NGS for 1 hour at room temperature. Nuclei were stained with TO-PRO™-3 Iodide (Invitrogen, UK) and mounted using ProLong Glass Antifade Mountant (Thermo Fisher, UK) using high precision coverslips (0.170 ± 0.01 mm thick; Roth, Germany, LH25.1). Densitometric analysis was used to assess the percentage area stained of immunoreactivity within the region of interest (ROI) (see Supplementary Methods 3). An adapted stereological method was used to estimate the number of sgACC neurones (see Supplementary Methods 4). Midbrain sections containing the VTA and SN at the insertion of the oculomotor nerve were used to estimate pigmented neurone numbers 27 . Estimation of dopaminergic and serotonergic fibre density For dopaminergic and serotonergic fibre analysis in sgACC, images were captured using a microscope with a motorised stage (Zeiss, Germany) coupled to a PC. Stereologer software (Stereologer, Bethesda, MD, USA) was used to ensure adequate and unbiased sampling. The ROI was drawn at 1.25X magnification and a randomly-oriented point grid superimposed over the image (Fig. 2 ). Within the ROI, 10–15 frames were captured at 10X magnification. DAT and serotonin transporter (5HTT) positive fibres within sgACC were assessed by counting the number of intersections between the linear probe and lines representing the surface feature within a dissector frame of known dimensions. The isotropic interaction between the linear probes and the surface feature was achieved by using VUR (vertical uniform random) sections in combination with cycloid sine-weighted line probes 28 . STED analysis Triple-colour STED microscopy was applied to assess DAT or 5HTT co-localisation with a presynaptic terminal marker (SNAP25) and phosphorylated α-synuclein (s129) which provides an indication of both physiological and pathological α-synuclein. STED 3D images were acquired using a Leica SP8 STED microscope and Application Suite X software (LAS X; Leica Microsystems) with 100x/1.4NA STED white oil immersion objective. Images of 256 × 256 pixels were obtained using 35x optical zoom, resulting in a pixel size of 13 × 13 nm. Images were deconvolved using Huygens Essential Software (Scientific Volume Imaging, Netherlands). The Object Analyser Advanced tool in Huygens was used to create 3D surfaces for each channel and obtain the quantitative measures of individual particles. Co-localisation measurements were used to assess spatial overlap between structures in different data channels. Synapses were defined by an overlap of greater than 80% of SNAP25 staining and DAT or 5HTT staining. Alpha-synuclein positive synapses were defined where s129 staining overlap with the synapse exceeded 50% of stained volume. Western Blot and Dot Blot analysis Western and dot blot analysis used established methods (see Supplementary Methods 5 and Supplementary table 1 ). Statistical analysis Statistical analyses were performed using SPSS Statistics version 22.0 (see Supplementary Methods 6). Results Pathology in Subgenual Cingulate A significant main effect of diagnosis on α-synuclein pathological burden was observed in sgACC H ( 3 ) = 31.370, p < 0.001, with significant increase identified in DLB cases overall, with or without depression, compared to controls ( p < 0.001; Fig. 1 B). Alpha-synuclein (5G4) pathological burden was significantly different in sgACC between groups H ( 3 ) = 40.174, p < 0.001, with significant increases in DLB cases overall, with or without depression compared to controls ( p < 0.001; Fig. 1 B). Tau pathological burden was also significantly different between groups H ( 3 ) = 12.140, p = 0.007. DLB cases with depression showed similar p-Tau burden compared to controls, however DLB cases overall ( p = 0.010) and DLB without depression ( p = 0.015) showed an elevated tau burden in sgACC compared to controls. Aβ burden was not significantly different between groups H ( 3 ) = 3.266, p = 0.352 (Fig. 1 B). Biochemical Analysis of α-synuclein Using s129 antibody to assess biochemical changes using sgACC fractionated tissue 29 generally supported immunohistochemical data, and showed no significant difference in α-synuclein levels between DLB cases with and without depression. Increased s129 was found in the crude sample (H ( 2 ) = 16.826, p < 0.001), supernatant ( H ( 2 ) = 7.115, p = 0.029), 0.1% Tween pellet ( H ( 2 ) = 11.988, p = 0.002), 0.1% Tween supernatant ( H ( 2 ) = 16.122, p < 0.001), 2% SDS pellet ( H ( 2 ) = 6.504, p = 0.039), 2% SDS supernatant ( H ( 2 ) = 9.159, p = 0.028) and urea fraction of tissue homogenates ( H ( 2 ) = 20.523, p < 0.001). DLB cases with depression showed higher s129 burden compared to controls in crude ( p = 0.001), supernatant ( p = 0.029), 0.1% Tween pellet ( p = 0.003), 0.1% Tween supernatant ( p < 0.001), 2% SDS supernatant ( p = 0.020) and urea ( p = 0.001). DLB cases without depression showed higher s129 levels compared to controls in the crude ( p = 0.002), 0.1% Tween pellet ( p = 0.045), 2% SDS pellet ( p = 0.010) and 2% SDS supernatant ( p = 0.035) and urea fraction (p < 0.001). There was no significant difference between DLB cases with or without depression in s129 immunoreactivity (Fig. 1 C). The neuronal specific marker (HuD) 30 showed no significant difference in neuronal density overall F ( 2 , 48 ) = 0.393, p = 0.677, or within layers II/III F ( 2 , 48 ) = 0.018, p = 0.982, or layer V F ( 2 , 48 ) = 1.183, p = 0.315 of the sgACC between groups (Fig. 1 D). Dopaminergic and Serotonergic Fibres in sgACC The sgACC receives dopaminergic projections from the VTA but also the SN 31–33 therefore we assessed pigmented dopaminergic neurones along with DAT and 5HTT positive fibres in the sgACC. VTA neurone number was significantly different between groups H ( 3 ) = 19.056, p < 0.001, with significantly lower neuronal count in DLB overall ( p = 0.001), DLB cases with depression ( p = 0.001) and DLB cases without depression compared to controls ( p = 0.035; Fig. 2 D). The number of neurones in the SN was also significantly different between the groups H ( 3 ) = 34.179, p < 0.001, with significantly fewer neurones observed in all groups compared to controls ( p < 0.001; Fig. 2 E). There were no significant differences in neurone counts between depressed and non-depressed donors in either the VTA or SN. DAT positive fibres in the sgACC differed significantly between the groups F ( 3 , 69 ) = 7.029, p < 0.001, with lower fibre density in DLB cases overall ( p = 0.004) and in DLB cases with depression compared to controls ( p < 0.001) but not in non-depressed DLB donors (Fig. 2 F). Since abnormal serotonergic neurotransmission is implicated in depression, serotonergic innervation of sgACC was assessed using an identical approach. Serotonergic fibres were more abundant in the sgACC compared to dopaminergic fibres χ2( 1 ) = 8.138, p = 0.004, however, no significant difference in the number of serotonergic fibres in the sgACC was observed between groups F ( 2 , 39 ) = 1.694, p = 0.197 (Fig. 2 G). No significant correlations occurred between dopaminergic neurones in VTA or SN, and DAT positive fibre density in sgACC within the groups (Supplementary Fig. 1). In the combined control and DLB groups however, a significant correlation was observed between DAT fibre density and VTA neurones r = 0.337, p = 0.007, as well as SN neurones r = 0.313, p = 0.011 (Fig. 2 H-I). Dopaminergic and serotonergic synapses in sgACC Morphometric findings were extended by assessment of serotonergic (5HTT and SNAP25 positive) and dopaminergic synapses (DAT and SNAP25 positive) in relation to phosphorylated α-synuclein (s129) in the sgACC using STED (Fig. 3 ). Due to the high resolution analysis provided by STED microscopy, an unbiased stereological analysis was not possible. No significant difference in the proportion of presynaptic serotonergic terminals F ( 2 , 22 ) = 1.258, p = 0.299 or the proportion of serotonergic synapses containing α-synuclein was observed between the groups F ( 2 , 22 ) = 0.057, p = 0.944. 5HTT containing synapses were not different in size between groups H ( 3 ) = 0.833, p = 0.842. No significant difference was observed in α-synuclein positive H ( 3 ) = 2.089, p = 0.554, or α-synuclein negative H ( 3 ) = 0.803, p = 0.749 serotonergic synapses between groups (Fig. 3 H, J, L). No significant differences were observed in DAT synapse proportion ( p = 0.582), or DAT synapses containing s129 between groups ( p = 0.823). DAT synapse volume was however, significantly different between groups H ( 3 ) = 12.007, p = 0.007, with larger DAT synapses observed in DLB overall ( p = 0.011), as well as non-depressed DLB cases ( p = 0.017) compared to control (Fig. 3 I). Specifically, DAT synapses containing s129 alpha-synuclein were larger compared to controls in the DLB group overall ( p = 0.043), and in the non-depressed DLB group ( p = 0.016), but not in the depressed DLB group. Alpha-synuclein negative dopaminergic synapses were significantly larger in DLB cases with depression ( p = 0.028) compared to control α-synuclein negative synapses (Fig. 3 K, M). Alpha-synuclein can interact with synaptic vesicle proteins 34 , therefore we determined if α-synuclein within dopaminergic or serotonergic synapses correlated with synaptic activity assessed using SNAP25. A significant positive correlation between the volume of s129 and SNAP25 within presynaptic 5HTT terminals was observed in DLB overall ( r = 0.288, p = 0.012), non-depressed DLB ( r = 0.313, p = 0.016) and DLB with depression ( r = 0.316, p = 0.030; Fig. 4 ). A significant positive correlation was observed between s129 volume and SNAP25 within DAT terminals in DLB cases with depression ( r = 0.379, p = 0.007; Fig. 4 ), but not in DLB cases without depression or controls. A significant correlation was observed between VTA neurones and the volume of DAT synapses in sgACC in DLB cases with depression ( r = 0.710, p = 0.049; Supplementary Fig. 2). Furthermore, a significant correlation in DLB cases with depression was observed between VTA neurone number and volume of α-synuclein positive DAT synapses ( r = 0.732, p = 0.016; Supplementary Fig. 3). No significant correlations were observed between SN neurones and DAT positive synapse volume in sgACC within groups (Supplementary Fig. 2). Monoaminergic Protein Analysis To determine the impact of DLB on dopaminergic and serotonergic markers we used western and dot blotting. DAT F ( 3 , 56 ) = 4.346, p = 0.008, tyrosine hydroxylase (TH) F ( 3 , 56 ) = 8.762, p < 0.001, dopamine decarboxylase (DDC) F ( 3 , 56 ) = 3.660, p = 0.018 and dopamine D3 receptor (D3DR) levels F ( 3 , 56 ) = 2.913, p = 0.042 were significantly different between the groups in the sgACC (Fig. 5 A-F). DLB cases overall showed significantly lower DAT ( p = 0.022), TH ( p = 0.002) and DDC levels ( p = 0.024) compared to controls. Significantly lower DAT ( p = 0.009), TH ( p = 0.003), DDC ( p = 0.041) and D3DR levels ( p = 0.049) were observed in DLB cases with depression compared to controls. DLB cases without depression showed significantly lower levels of TH ( p = 0.012) compared to controls. No significant difference was observed in dopamine D2 receptors (D2DR) F ( 3 , 56 ) = 0.620, p = 0.605 or D4 receptors (D4DR) F ( 3 , 56 ) = 2.115, p = 0.109 between groups. No significant changes in 5HTT F ( 3 , 56 ) = 0.064, p = 0.978, tryptophan hydroxylase 2 (TPH-2) F ( 3 , 56 ) = 1.719, p = 0.173, 5HT1 A receptor F ( 3 , 56 ) = 0.258, p = 0.855 or 5HT3 B receptor F ( 3 , 56 ) = 2.156, p = 0.062 was observed between the groups (Fig. 5 H-L). A significant difference in 5HT2 A receptor protein levels was observed in the sgACC between groups F ( 2 , 34 ) = 3.858, p = 0.019, with significantly lower 5HT2 A observed in DLB cases overall ( p = 0.026) and in DLB cases with depression compared to controls ( p = 0.044). Heat map analysis showed no clear separation of disease groups based on monoaminergic proteins (Fig. 6 ). Using linear discriminant analysis to separate cases based on monoaminergic markers to determine the greatest influence on depression showed DAT (Wilks’ Lambda, 0.535, p = 0.0004) and D4DR (Wilks’ Lambda 0.442, p = 0.0002) in combination had greatest predictive values, showing 78% accuracy in re-classifying the original data. Discussion DLB patients are at high risk of developing major depression, with depression having a significant impact on quality of life 3 . Depression in DLB correlates with α-synuclein pathology in late onset MDD, indicating that α-synuclein pathology may be a factor in MDD development in older individuals 35 . The sgACC is an area intimately involved in the aetiology of depression, with structural and functional changes in early onset MDD 10, 26 and a high α-synuclein pathological burden in DLB 27 . Despite high levels of α-synuclein in the sgACC, we found no significant difference between depressed and non-depressed DLB donors for α-synuclein pathology burden suggesting gross pathology within the sgACC is not a significant driver of depressive symptoms. Significant neurone loss within the sgACC in DLB is not apparent, and there is no association with depression. Although the basis for depression in DLB is complex, our results indicate reduced dopaminergic innervation in sgACC with changes in dopaminergic synaptic function and decreased levels of dopaminergic markers contributing. This may warrant clinical trials of D2 dopamine receptor agonists since pramipexole and pergolide show efficacy in reducing depression in patients with PD 36, 37 , as well as symptoms of major depression in patients without PD 38, 39 . The mesolimbic dopamine pathway from the VTA to cortical and subcortical regions includes the cingulate with additional projections from the dorsal tier of the SN 33 . VTA dopaminergic neurones play a role in reward and stress 20 with dopaminergic dysfunction in the mesolimbic system contributing to anhedonia and loss of motivation in MDD 21 . We identified a decrease in VTA cell number in DLB cases overall, but no significant loss in relation to depression in DLB. Compared to the major loss of neurones in the SN 27 , the VTA shows relative preservation of neurones in PD 27, 40 . Similar to the current findings, severe cell loss and gliosis is associated with the presence of depression in PD 41 , with VTA neuronal density having a significant effect 42 . In PD, PDD, and DLB, higher midbrain α-synuclein burden associates with depressive symptoms 27 suggesting that mesolimbic and mesocortical projections rather than nigrostriatal changes contribute to depression in DLB, similar to PD 43 . In this study, we observed reduced sgACC dopaminergic fibre density in DLB cases with depression as a potential indicator of the reduced VTA innervation. In PD, motor symptoms appear when around 50% of SN dopaminergic neurones are lost 44 , although striatal innervation at onset of motor symptoms is more severe, showing around 80% terminal loss 45 . Dopaminergic axons and terminals are the main site of pathology, with the neurodegenerative process in PD thought to follow a retrograde pathway originating in the striatal terminals 46 . The enhanced dopaminergic fibre loss seen in this study in DLB cases with depression in the absence of severe (~ 20%) VTA neuronal loss may represent a similar effect with axon and terminal loss prior to major cell death. Our STED analysis showed phosphorylated α-synuclein within sgACC dopaminergic synapses and increased phosphorylated α-synuclein in sgACC tissue homogenates. In depressed DLB cases, elevated synaptic α-synuclein corresponded with elevated SNAP25, a finding also seen with serotonergic terminals. One possibility is that pathological α-synuclein sequesters SNAP25 within synapses leading to synaptic dysfunction. Alpha-synuclein modulates synaptic activity through effects on vesicle recycling and release by assisting with SNARE complex formation 34, 47 . The positive correlation identified in the current study may represent a synaptic response to increase activity and maintain normal neurotransmission following reduction in cell numbers 48 . Pathological fibrillar α-synuclein can however, rapidly promote aberrant synaptic activity following neuronal application 49 . In forming aggregates within synapses, fibrillar α-synuclein may cause an effective depletion of functional SNARE proteins including SNAP25, reducing the effective synaptic vesicle pool. Fibrillar and oligomeric α-synuclein also has direct effects on synaptic machinery by depleting SNARE complexes 50 . This may underscore the increase in SNAP25 within α-synuclein containing dopaminergic synapses, with SNAP25 increased, but non-functional due to sequestration in α-synuclein aggregates 51 . The increased volumes of α-synuclein negative synapses in depressed DLB donors may be a compensatory response to reduced function of α-synuclein containing synapses in depressed cases. The combined effects of dopaminergic synapse reduction due to cell loss and reduced synaptic efficiency in remaining synapses may cause an effective depletion of dopamine to the sgACC cortex and contribute to depressive symptoms. In this study, we found a loss of dopaminergic proteins in DLB cases with depression compared to controls, including DAT, TH and DDC. Using hierarchical clustering, DAT and DRD4 showed significant associations with depression in DLB. These reductions align with DAT fibre loss in depressed DLB cases and reinforce the general loss of dopaminergic innervation in DLB, but particularly in DLB experiencing depression 25, 27 . Dopaminergic deficits including reduced striatal DAT binding has been observed in depressed patients with anhedonia 22 and additionally in PD and DLB, with DLB cases showing greater DAT loss in caudate compared to PD 52 , with reduced ACC DAT binding in DLB 53 . The use of dopamine agonists in MDD can have significant benefits, and may be beneficial in DLB if suitable treatment regimens are identified 54, 55 . Our results indicate minimal changes in post-synaptic D2DR in DLB cases. The highest levels of D2DR are found in the striatum, nucleus accumbens and olfactory tubercle, with significant levels in the SN, VTA, amygdala, hippocampus and cortex 56 . In PD, SN neurone loss results in reduced striatal dopamine with increased striatal D2DR as a compensatory mechanism in response to low dopamine 57 . Increased dopamine D2/D3 receptor binding and lower DAT activity has also been shown in MDD, potentially reflecting compensatory changes 58, 59 . This does not appear to occur in DLB however, where reduced striatal and cortical D2DR occurs 60, 61 . D3DR are expressed pre- and postsynaptically and have the highest affinity for dopamine 62 . DRD3 is a clinically relevant treatment target following identification of DRD3 downregulation in MDD 63 . A reduction in DRD3 in the ventral striatum has been observed in PD cases 64 , but with either downregulation 65 or no change 60 in DLB. Our results indicate that reduced DRD3 levels may play a role in depression in DLB, and DRD3 specific dopaminergic agents may be beneficial. Pramipexole and ropinirole improve depressive symptoms in PD and may provide benefit for depression in DLB, although careful monitoring may be needed to prevent unwanted side effects including impulse control disorders and hallucinations 54, 66, 67 . In PD, improved working and episodic memory occurs with dopamine agonists, indicating that dopaminergic agonists may additionally improve cognition 68, 69 . Our findings show no significant changes in sgACC DRD4 levels in DLB despite an association based on hierarchical clustering. DRD4 shows the lowest levels of expression of dopamine receptors in the brain, with highest densities in anterior limbic and cortical forebrain suggesting cognitive, executive, and motivational regulation 70 . Similar to previous studies indicating that DRD4 are not implicated in the modulation of depressive-like behaviours 71 , our findings suggest that DRD4 may not be a treatment target. In contrast to dopaminergic changes, serotonergic involvement in depression in DLB was limited, comparable with similar studies 42, 72 . Whilst the dorsal raphe nucleus (DRN) shows LB pathology in DLB and PD, studies of neurone loss are variable with reduction 73 or no change in DRN neurones in PD or PDD 72, 74 . In one report in DLB, neuronal loss is present within both the DRN and median raphe 75 . In the current study, due to the tissue sampling strategy and differences in DRN projection sites (Ren et al., 2018), DRN neurone numbers were not assessed. We however found no change in sgACC serotonergic fibre density in DLB similar to findings in the amygdala and dorsal prefrontal cortex in LBD donors with and without depression 72 . We did however find α-synuclein associated with serotonergic synapses and a positive correlation between SNAP25 volume and α-synuclein volume. This finding in 5HT synapses may indicate 5HT terminal dysfunction without a change in innervation in the sgACC since we saw no change in fibre density or 5HTT protein levels. Other studies have also showed no association with 5HTT levels with depression or anxiety in PD 76 . Future studies should however determine the effects of α-synuclein pathology in DLB in the brainstem and midbrain serotonergic system using appropriate stereological approaches. There has been considerable in vivo use of 5HTT ligands in PD and to a lesser extent DLB. In drug naïve PD, there were no changes in non-apathetic PD but reductions in [( 11 )C-N,N-dimethyl-2-(-2-amino-4-cyanophenylthio)-benzylamine (DASB)] retention in apathetic patients in the sgACC when compared to non-apathetic PD suggesting reduced serotonergic innervation 77 . Similarly, reduced SERT assessed with DASB is seen in caudate and ACC in early stage PD and throughout the disease course 78–80 and at post mortem 81 . In depressed PD patients however, increased DASB retention was observed in several cortical areas although more generally there was no change in DASB retention in ACC in MDD 82, 83 . In vivo studies in DLB have used the combined DAT and SERT ligand 123 I-N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) nortropane ( 123 I-FP-CIT) where the assumption is that cortical retention only represents 5HT innervation despite DA terminals being present. Studies with 123 I-FP-CIT have shown either reduced cortical SERT 84–86 , or no cortical changes 87 . No reduction in SERT was seen in the ACC at post mortem in PDD, although brain regions such as caudate showed reductions 88 . Similarly, in DLB compared to control or non-depressed patients, SERT assessed with cyanoimipramine showed preserved cortical binding in depressed DLB donors 89, 90 . Whilst these prior studies have shown variable outcomes, our findings indicate SERT reductions in DLB are mild or absent and contributions to development of depression are limited. Decreased serotonin 5HT1 A binding is inidcated in the ACC in MDD patients using PET 14 however, we observed no alteration of 5HT1 A protein levels in DLB. This contrasts with findings using the 5HT1a ligand 8-Hydroxy-2-Dipropylaminotetralin showing increased binding but altered K D in DLB and PD 91 . This may suggest a change in receptor occupancy due to changes in protein conformation but not protein levels. However, our finding of decreased 5HT2 A protein in sgACC in DLB donors with depression corresponds with reduced 5HT2 A binding in ACC and DLPFC in MDD 15 and reduced [ 3 H]-ketanserin binding in DLB and PDD 92, 93 . These changes in 5HT2 A but not 5HT1 A suggests region specific serotonergic receptor changes that may benefit from selective targeting. Pimavanserin, a 5HT2a selective inverse agonist and antagonist, has been used in psychosis treatment in PD 94, 95 . Despite mixed benefits in MDD 96, 97 , an open label study of Pimavanserin for depression in PD in the absence of psychosis showed delayed benefit in an 8-week trial with 60% of patients showing improved mood 98 although longer-term use of Pimavanserin may show reduced efficacy 99 . The possibility of using Pimavanserin in the treatment of depression in may be warranted in certain DLB patients 100 . In summary, these results suggest that there is primarily a reduced dopaminergic innervation in the sgACC in DLB, along with reduced levels of dopaminergic markers and receptors, and changes in dopaminergic synaptic function. Careful treatment with selective dopaminergic agonists or positive allosteric modulators may be beneficial in alleviating depressive symptoms in DLB. Declarations Conflict of Interest The authors declare no competing interests. The views expressed are those of the author(s) and not necessarily those of the NHS, NIHR or the UK Department of Health. Acknowledgements This project is supported by a studentship through the Alzheimer’s Society Doctoral Training Centre. Tissue for this study was provided by the Newcastle Brain Tissue Resource, which is funded in part by a grant from the UK Medical Research Council and the Brains for Dementia research, a joint venture between Alzheimer’s Society and Alzheimer’s Research UK. The research was also partly funded by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre based at Newcastle Hospitals NHS Foundation Trust and Newcastle University. References McKeith IG, Dickson DW, Lowe J, Emre M, O'Brien JT, Feldman H et al. Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium. Neurology 2005; 65(12): 1863–1872. McAleese KE, Colloby S, Attems J, Thomas A, Francis PT. 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Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files SupplementaryFigures.docx SupplementaryTables.docx SupplementaryMethods.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3953937","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":279416903,"identity":"0cb98181-7378-4719-b28a-ee7481c0343a","order_by":0,"name":"Lina Gliaudelytė","email":"data:image/png;base64,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","orcid":"","institution":"Newcastle University","correspondingAuthor":true,"prefix":"","firstName":"Lina","middleName":"","lastName":"Gliaudelytė","suffix":""},{"id":279416904,"identity":"9f79fd5a-cf4d-4325-96f3-49a5f82b466b","order_by":1,"name":"Steven Rushton","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"","lastName":"Rushton","suffix":""},{"id":279416905,"identity":"0813854f-4909-4a54-b20f-a91cbad3a3b0","order_by":2,"name":"Alan Thomas","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Alan","middleName":"","lastName":"Thomas","suffix":""},{"id":279416906,"identity":"f418fb38-610a-4b08-9599-5f7443286f04","order_by":3,"name":"Rolando Berlinguer Palmini","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rolando","middleName":"Berlinguer","lastName":"Palmini","suffix":""},{"id":279416907,"identity":"c71061b2-c945-43b2-ae36-5c7b861126c9","order_by":4,"name":"Christopher Morris","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Morris","suffix":""}],"badges":[],"createdAt":"2024-02-13 16:25:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3953937/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3953937/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52918148,"identity":"1265339f-5216-476b-abe8-c24416501746","added_by":"auto","created_at":"2024-03-18 17:06:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":650713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Pathology on the sgACC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e Photomicrographs of α-synuclein (KM51), α-synuclein (5G4), Tau (AT8) and Aβ (4G8) pathology in sgACC (BA25) in controls, DLB cases with and without depression. Magnification x10; scale bars represent 100μm. \u003cstrong\u003eB) \u003c/strong\u003eα-synuclein (KM51), (5G4), Tau (AT8) and Aβ (4G8) pathology (% area stained) in sgACC in Controls, DLB cases overall, with and without depression; (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 and *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, compared to control group). \u003cstrong\u003eC) \u003c/strong\u003eDot blotting of α-synuclein phosphorylated at position serine 129 in sgACC within different tissue fractions: crude, supernatant (cytoplasmic soluble proteins), 0.1% Tween pellet (soluble membrane bound proteins), 0.1% Tween supernatant (Tween soluble membrane proteins), 2% SDS pellet (insoluble membrane bound proteins), 2% SDS supernatant (SDS soluble membrane proteins) and 6M Urea supernatant (highly insoluble proteins). \u003cstrong\u003eD)\u003c/strong\u003e Neurones immunopositive for the neuronal marker HuD were determined in layers II/III and layer V and overall (cells per/mm\u003csup\u003e2\u003c/sup\u003e) within sgACC.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/66e43687149fecb1bd4960b9.png"},{"id":52918144,"identity":"93f4a03a-4fb6-4e83-9811-6076c0709612","added_by":"auto","created_at":"2024-03-18 17:06:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":543988,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of Dopaminergic cells in Ventral Tegmental Area and Substantia Nigra, and Dopaminergic and Serotonergic fibres in sgACC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003eFor stereological imaging, a region of interest was drawn at 1.25X and a point grid was superimposed over the area, with green points representing the coordinates of sampling; \u003cstrong\u003eB)\u003c/strong\u003e Randomly sampled frames in the x-y axis were taken at 63X, with neurones counted within a dissector frame of known dimensions to allow for an estimation of neurone numbers;\u003cstrong\u003e C) \u003c/strong\u003eFor the estimation of monoaminergic fibres within the outlined reference space, the fibres intersecting (marked green) a grid of randomly orientated cycloids were counted; \u003cstrong\u003eD) \u003c/strong\u003epigmented dopaminergic neurones in the VTA; \u003cstrong\u003eE)\u003c/strong\u003e pigmented dopaminergic neurones in the SN; \u003cstrong\u003eF)\u003c/strong\u003eDopaminergic fibres (DAT positive) in sgACC; \u003cstrong\u003eG)\u003c/strong\u003e Serotonergic fibres (5HTT positive) in sgACC were assessed in controls, DLB cases overall, DLB cases without (DLB-D) and DLB cases with depression (DLB+D) (Significant at *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 and ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001); \u003cstrong\u003eH)\u003c/strong\u003e Correlation analysis between dopaminergic neurones in ventral tegmental area (VTA)\u003cstrong\u003e \u003c/strong\u003e(\u003cem\u003er\u003c/em\u003e=0.337, \u003cem\u003ep\u003c/em\u003e=0.007), and \u003cstrong\u003eI)\u003c/strong\u003e substantia nigra (SN) (\u003cem\u003er\u003c/em\u003e=0.313,\u003cem\u003e p\u003c/em\u003e=0.011) with DAT positive fibres in sgACC.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/1520c8aa2c8d0d45f92676cd.png"},{"id":52918147,"identity":"a327a6a1-919c-4643-8a5d-fe489fd003fd","added_by":"auto","created_at":"2024-03-18 17:06:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":413926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStimulated Emission Depletion (STED) imaging of Dopaminergic and Serotonergic Synapses in Subgenual anterior cingulate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA) - \u003c/strong\u003eSTED 3D image of dopaminergic fibres and terminals (DAT; green), presynaptic terminals (SNAP25; yellow) and phosphorylated α-synuclein (s129; red); \u003cstrong\u003eB)\u003c/strong\u003e \u003cstrong\u003e-\u003c/strong\u003eDeconvolved STED image; \u003cstrong\u003eC,\u003c/strong\u003e \u003cstrong\u003eE) -\u003c/strong\u003e 3D surfaces of dopaminergic fibres and presynaptic terminals; \u003cstrong\u003eD\u003c/strong\u003e,\u003cstrong\u003e F)\u003c/strong\u003e \u003cstrong\u003e- \u003c/strong\u003e3D surfaces of presynaptic terminals and α-synuclein; \u003cstrong\u003eG)\u003c/strong\u003e \u003cstrong\u003e- \u003c/strong\u003e3D surfaces of co-localisation of all 3 channels; \u003cstrong\u003eH)\u003c/strong\u003e Co-localisation of serotonergic (5HTT +ve) and \u003cstrong\u003eI)\u003c/strong\u003e dopaminergic (DAT +ve) synapses with pre-synaptic terminal marker (SNAP25) (\u0026gt;80% co-localisation); \u003cstrong\u003eJ, K)\u003c/strong\u003e Serotonergic and dopaminergic synapses α-synuclein (s129) +ve, and \u003cstrong\u003eL, M)\u003c/strong\u003e α-synuclein (s129)–ve synapses in sgACC were assessed in controls, DLB cases overall, DLB cases without (DLB-D) and DLB cases with depression (DLB+D) (Significant at *\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/67ff708822507445714fad8d.png"},{"id":52918141,"identity":"c9fefe68-fe7e-4927-b441-e1822ec1fde7","added_by":"auto","created_at":"2024-03-18 17:06:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":376148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of phosphorylated α-synuclein (s129) on serotonergic and dopaminergic synapses.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTop)\u003c/strong\u003eThe relationship between 5HTT and SNAP25, and \u003cstrong\u003eBottom) \u003c/strong\u003eDAT and SNAP25 with s129 within synapses in the sgACC was assessed using STED microscopy and Spearman’s correlation analysis in controls, DLB cases overall, DLB cases with (DLB+D) and without (DLB-D) depression.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/40e0757ce4d16546dd30fcf5.png"},{"id":52918146,"identity":"f68f454c-dfde-4694-8b25-353f0124b002","added_by":"auto","created_at":"2024-03-18 17:06:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":192707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDot blot analysis of dopaminergic and serotonergic markers and receptors in sgACC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDopaminergic markers, including \u003cstrong\u003eA\u003c/strong\u003e) tyrosine hydroxylase (TH), \u003cstrong\u003eB\u003c/strong\u003e) dopamine transporter (DAT), \u003cstrong\u003eC)\u003c/strong\u003e dopamine decarboxylase (DDC), and \u003cstrong\u003eD-E)\u003c/strong\u003edopamine receptors D2, D3 and D4, as well as serotonergic markers, including \u003cstrong\u003eH) \u003c/strong\u003eserotonin transporter (5HTT), \u003cstrong\u003eB)\u003c/strong\u003e tryptophan hydroxylase-2 (TPH-2), and \u003cstrong\u003eJ-L)\u003c/strong\u003e serotonin receptors 5HT1A, 5HT2A and 5HT3B were assessed in sgACC in age matched controls, DLB cases overall, DLB cases with (DLB+D) and without (DLB-D) depression; (*\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05 and ***\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001, compared to appropriate group).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/bbc5bb0f83e6039abef811cf.png"},{"id":52918143,"identity":"aa32cc97-ebc3-4b3b-b467-04ed5f534077","added_by":"auto","created_at":"2024-03-18 17:06:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":112381,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeat map analysis of monoaminergic markers and receptors in the sgACC.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHierarchical clustering analysis was performed based on monoaminergic protein relative expression in sgACC in controls, DLB cases with (DLB+D) and without (DLB-D) depression.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/68299d60c6cc6d52453990dd.png"},{"id":77820005,"identity":"47aee225-29c7-467e-bfa9-e06cd5e5228e","added_by":"auto","created_at":"2025-03-05 19:58:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3663364,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/1f5ff321-78ef-4018-9cb1-a702ee57b512.pdf"},{"id":52918140,"identity":"8f46d34d-da0a-4f9f-9c7d-adcaf158de8d","added_by":"auto","created_at":"2024-03-18 17:06:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":106503,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/5d213f0a95bc444597724fa1.docx"},{"id":52918139,"identity":"13bf7d88-a471-4348-922f-64c5d0d97c14","added_by":"auto","created_at":"2024-03-18 17:06:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":123415,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/84c4a57938ea100c14164bd6.docx"},{"id":52918145,"identity":"bc87e48c-8b51-4d48-996b-6b1904f3a8e6","added_by":"auto","created_at":"2024-03-18 17:06:07","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":31887,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-3953937/v1/90db545b3c4adda0bc762c22.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Dopaminergic Changes in the Subgenual Cingulate Cortex in Dementia with Lewy Bodies Associates with Presence of Depression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDementia with Lewy bodies (DLB) is a significant cause of morbidity in older populations, representing between 5\u0026ndash;20% of all clinical dementia cases \u003csup\u003e1, 2\u003c/sup\u003e. DLB shows core symptoms of fluctuating cognition, parkinsonism, the presence of recurrent complex visual hallucinations and REM sleep behaviour disorder \u003csup\u003e3\u003c/sup\u003e. Psychiatric features are highly prevalent in many DLB patients, with major depression present in 50\u0026ndash;80% of cases \u003csup\u003e4\u003c/sup\u003e. Depression is frequently present at prodromal DLB stages, persisting throughout the clinical course \u003csup\u003e5\u003c/sup\u003e. Depression in dementia is associated with poor quality of life, increased morbidity and more rapid cognitive decline, and treatment would have significant patient benefit \u003csup\u003e6, 7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe subgenual anterior cingulate cortex (sgACC) is a key brain region associated with mood and anxiety, and relays emotional information between limbic, cortical and subcortical regions \u003csup\u003e8, 9\u003c/sup\u003e. Abnormalities in sgACC activity, connectivity and grey matter volume have been shown in depressive disorders \u003csup\u003e10\u003c/sup\u003e, with sgACC used as a target for treating treatment resistant depression with deep brain stimulation \u003csup\u003e11, 12\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe monoamine hypothesis of depression in unipolar major depressive disorder (MDD), suggests that reduction in serotonergic and noradrenergic neurotransmission underpins depressive symptoms \u003csup\u003e13\u003c/sup\u003e. Considerable evidence supports the monoamine hypothesis in individuals with MDD, with reduced 5HT concentrations observed in serum and reduced 5HIAA in CSF in patients, along with decreased serotonin 5HT1\u003csub\u003eA\u003c/sub\u003e and 5HT2\u003csub\u003eA\u003c/sub\u003e binding in anterior cingulate cortex (ACC) in MDD \u003csup\u003e14, 15\u003c/sup\u003e. However, reduced levels of 5HT as a basis of depression have been challenged by some, primarily based on reduced and delayed response to 5HT based therapies \u003csup\u003e16, 17\u003c/sup\u003e. Data on serotonergic treatment of depression in DLB is lacking, and in depression in Parkinson\u0026rsquo;s disease (PD) serotonergic treatment shows minimal effects in a few small-scale trials \u003csup\u003e18, 19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDopamine plays a role in reward and stress \u003csup\u003e20\u003c/sup\u003e with dysfunction of dopaminergic neurotransmission within the mesolimbic and mesocortical systems contributing to anhedonia and loss of motivation in depression \u003csup\u003e21\u003c/sup\u003e. A significant reduction in dopamine transporter (DAT) uptake is observed in anhedonic depressed patients \u003csup\u003e22\u003c/sup\u003e, along with increased striatal D2 receptor binding in MDD suggesting decreased dopamine turnover \u003csup\u003e23\u003c/sup\u003e. Depression in PD is associated with nigral and mesolimbic dopaminergic pathway dysfunction with reduced ventral tegmental area (VTA), cingulate and amygdala volumes on MRI, and reduced [11C]RTI-32 DAT uptake in limbic regions in PD patients, with depression correlated with loss of dopamine projections from the VTA \u003csup\u003e24\u003c/sup\u003e and substantia nigra (SN) \u003csup\u003e25\u003c/sup\u003e. Whilst PD with dementia shows reduced VTA neurone numbers, altered VTA neurone number in DLB is mild although neurone dysfunction has not been established (Patterson et al 2018). However, both DLB and PD show SN dopaminergic neurone loss and the contribution of SN dopamine depletion is unknown (Patterson et al 2018). What role dopaminergic systems plays in DLB patients with depression is unclear.\u003c/p\u003e \u003cp\u003eIn this study, we investigated dopaminergic parameters in relation to depression in DLB. We focussed on the subgenual anterior cingulate cortex (sgACC) due to its important role in mood regulation and the symptomatic expression of depression, and as a region vulnerable to α-synuclein pathology in DLB \u003csup\u003e10, 26\u003c/sup\u003e. Little is known about how changes in dopaminergic neurotransmission in the sgACC might relate to development of depression in DLB, despite high levels of α-synuclein and other neurodegenerative pathology within the sgACC \u003csup\u003e27\u003c/sup\u003e. We therefore assessed dopaminergic innervation in the sgACC using a combination of stereology, high resolution stimulated emission depletion (STED) microscopy, and protein determination using post-mortem tissue samples from DLB cases with and without depression to investigate associations with depression.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eClinical Information\u003c/h2\u003e \u003cp\u003ePost-mortem tissue was obtained from the Newcastle Brain Tissue Resource (NBTR). Ethical approval was granted by Newcastle and North Tyneside-1 National Health Service (NHS) Research Ethics Committee. Donors had received clinical assessments during life and consented to the use of brain tissue for research purposes. Seventeen controls, 15 DLB cases without depression and 13 DLB cases with depression were included, with 12 from each group used for biochemical analysis. Inclusion criteria for depression diagnosis used the Cornell Scale for Depression in Dementia (CSDD) (score\u0026thinsp;\u0026ge;\u0026thinsp;8) as a validated rating scale. Alternatively, the Geriatric Depression Scale (GDS) (score\u0026thinsp;\u0026ge;\u0026thinsp;10) was used if CSDD scores were not available (see Supplementary Methods 1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eFormalin fixed paraffin embedded tissue blocks from the right hemisphere containing the sgACC (Brodmann area: BA25) sampled at the rostrum of the corpus callosum were cut at 10\u0026micro;m using a rotary microtome. Immunohistochemical staining used established protocols (see Supplementary Methods 2).\u003c/p\u003e \u003cp\u003eFor STED microscopy, sections were blocked using 10% goat serum (NGS: Sigma G9023) for one hour at room temperature, followed by incubation with primary antibodies overnight at 4\u0026deg;C. After washes in TBS, sections were incubated with secondary fluorescent antibodies in TBS and 10% NGS for 1 hour at room temperature. Nuclei were stained with TO-PRO\u0026trade;-3 Iodide (Invitrogen, UK) and mounted using ProLong Glass Antifade Mountant (Thermo Fisher, UK) using high precision coverslips (0.170\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 mm thick; Roth, Germany, LH25.1).\u003c/p\u003e \u003cp\u003eDensitometric analysis was used to assess the percentage area stained of immunoreactivity within the region of interest (ROI) (see Supplementary Methods 3). An adapted stereological method was used to estimate the number of sgACC neurones (see Supplementary Methods 4). Midbrain sections containing the VTA and SN at the insertion of the oculomotor nerve were used to estimate pigmented neurone numbers \u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of dopaminergic and serotonergic fibre density\u003c/h2\u003e \u003cp\u003eFor dopaminergic and serotonergic fibre analysis in sgACC, images were captured using a microscope with a motorised stage (Zeiss, Germany) coupled to a PC. Stereologer software (Stereologer, Bethesda, MD, USA) was used to ensure adequate and unbiased sampling. The ROI was drawn at 1.25X magnification and a randomly-oriented point grid superimposed over the image (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Within the ROI, 10\u0026ndash;15 frames were captured at 10X magnification. DAT and serotonin transporter (5HTT) positive fibres within sgACC were assessed by counting the number of intersections between the linear probe and lines representing the surface feature within a dissector frame of known dimensions. The isotropic interaction between the linear probes and the surface feature was achieved by using VUR (vertical uniform random) sections in combination with cycloid sine-weighted line probes \u003csup\u003e28\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSTED analysis\u003c/h2\u003e \u003cp\u003eTriple-colour STED microscopy was applied to assess DAT or 5HTT co-localisation with a presynaptic terminal marker (SNAP25) and phosphorylated α-synuclein (s129) which provides an indication of both physiological and pathological α-synuclein. STED 3D images were acquired using a Leica SP8 STED microscope and Application Suite X software (LAS X; Leica Microsystems) with 100x/1.4NA STED white oil immersion objective. Images of 256 \u0026times; 256 pixels were obtained using 35x optical zoom, resulting in a pixel size of 13 \u0026times; 13 nm. Images were deconvolved using Huygens Essential Software (Scientific Volume Imaging, Netherlands). The Object Analyser Advanced tool in Huygens was used to create 3D surfaces for each channel and obtain the quantitative measures of individual particles. Co-localisation measurements were used to assess spatial overlap between structures in different data channels. Synapses were defined by an overlap of greater than 80% of SNAP25 staining and DAT or 5HTT staining. Alpha-synuclein positive synapses were defined where s129 staining overlap with the synapse exceeded 50% of stained volume.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot and Dot Blot analysis\u003c/h2\u003e \u003cp\u003eWestern and dot blot analysis used established methods (see Supplementary Methods 5 and Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS Statistics version 22.0 (see Supplementary Methods 6).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePathology in Subgenual Cingulate\u003c/h2\u003e \u003cp\u003eA significant main effect of diagnosis on α-synuclein pathological burden was observed in sgACC \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;31.370, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, with significant increase identified in DLB cases overall, with or without depression, compared to controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Alpha-synuclein (5G4) pathological burden was significantly different in sgACC between groups \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;40.174, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, with significant increases in DLB cases overall, with or without depression compared to controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Tau pathological burden was also significantly different between groups \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;12.140, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007. DLB cases with depression showed similar p-Tau burden compared to controls, however DLB cases overall (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010) and DLB without depression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.015) showed an elevated tau burden in sgACC compared to controls. Aβ burden was not significantly different between groups \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.266, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.352 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical Analysis of α-synuclein\u003c/h2\u003e \u003cp\u003eUsing s129 antibody to assess biochemical changes using sgACC fractionated tissue \u003csup\u003e29\u003c/sup\u003e generally supported immunohistochemical data, and showed no significant difference in α-synuclein levels between DLB cases with and without depression. Increased s129 was found in the crude sample \u003cem\u003e(H\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;16.826, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), supernatant (\u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;7.115, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029), 0.1% Tween pellet (\u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;11.988, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), 0.1% Tween supernatant (\u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;16.122, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 2% SDS pellet (\u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;6.504, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.039), 2% SDS supernatant (\u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;9.159, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) and urea fraction of tissue homogenates (\u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;20.523, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). DLB cases with depression showed higher s129 burden compared to controls in crude (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), supernatant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.029), 0.1% Tween pellet (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003), 0.1% Tween supernatant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), 2% SDS supernatant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020) and urea (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). DLB cases without depression showed higher s129 levels compared to controls in the crude (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002), 0.1% Tween pellet (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.045), 2% SDS pellet (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010) and 2% SDS supernatant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035) and urea fraction (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was no significant difference between DLB cases with or without depression in s129 immunoreactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eThe neuronal specific marker (HuD) \u003csup\u003e30\u003c/sup\u003e showed no significant difference in neuronal density overall \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.393, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.677, or within layers II/III \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.018, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.982, or layer V \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;1.183, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.315 of the sgACC between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDopaminergic and Serotonergic Fibres in sgACC\u003c/h2\u003e \u003cp\u003eThe sgACC receives dopaminergic projections from the VTA but also the SN \u003csup\u003e31\u0026ndash;33\u003c/sup\u003e therefore we assessed pigmented dopaminergic neurones along with DAT and 5HTT positive fibres in the sgACC. VTA neurone number was significantly different between groups \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;19.056, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, with significantly lower neuronal count in DLB overall (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001), DLB cases with depression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and DLB cases without depression compared to controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.035; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The number of neurones in the SN was also significantly different between the groups \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;34.179, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, with significantly fewer neurones observed in all groups compared to controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). There were no significant differences in neurone counts between depressed and non-depressed donors in either the VTA or SN.\u003c/p\u003e \u003cp\u003eDAT positive fibres in the sgACC differed significantly between the groups \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;7.029, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, with lower fibre density in DLB cases overall (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004) and in DLB cases with depression compared to controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not in non-depressed DLB donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Since abnormal serotonergic neurotransmission is implicated in depression, serotonergic innervation of sgACC was assessed using an identical approach. Serotonergic fibres were more abundant in the sgACC compared to dopaminergic fibres χ2(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;8.138, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004, however, no significant difference in the number of serotonergic fibres in the sgACC was observed between groups \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;1.694, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.197 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). No significant correlations occurred between dopaminergic neurones in VTA or SN, and DAT positive fibre density in sgACC within the groups (Supplementary Fig.\u0026nbsp;1). In the combined control and DLB groups however, a significant correlation was observed between DAT fibre density and VTA neurones \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.337, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007, as well as SN neurones \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.313, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDopaminergic and serotonergic synapses in sgACC\u003c/h2\u003e \u003cp\u003eMorphometric findings were extended by assessment of serotonergic (5HTT and SNAP25 positive) and dopaminergic synapses (DAT and SNAP25 positive) in relation to phosphorylated α-synuclein (s129) in the sgACC using STED (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Due to the high resolution analysis provided by STED microscopy, an unbiased stereological analysis was not possible.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNo significant difference in the proportion of presynaptic serotonergic terminals \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;1.258, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.299 or the proportion of serotonergic synapses containing α-synuclein was observed between the groups \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.057, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.944. 5HTT containing synapses were not different in size between groups \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.833, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.842. No significant difference was observed in α-synuclein positive \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.089, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.554, or α-synuclein negative \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.803, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.749 serotonergic synapses between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, J, L).\u003c/p\u003e \u003cp\u003eNo significant differences were observed in DAT synapse proportion (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.582), or DAT synapses containing s129 between groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.823). DAT synapse volume was however, significantly different between groups \u003cem\u003eH\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;12.007, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007, with larger DAT synapses observed in DLB overall (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.011), as well as non-depressed DLB cases (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017) compared to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). Specifically, DAT synapses containing s129 alpha-synuclein were larger compared to controls in the DLB group overall (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.043), and in the non-depressed DLB group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016), but not in the depressed DLB group. Alpha-synuclein negative dopaminergic synapses were significantly larger in DLB cases with depression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) compared to control α-synuclein negative synapses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, M).\u003c/p\u003e \u003cp\u003eAlpha-synuclein can interact with synaptic vesicle proteins \u003csup\u003e34\u003c/sup\u003e, therefore we determined if α-synuclein within dopaminergic or serotonergic synapses correlated with synaptic activity assessed using SNAP25. A significant positive correlation between the volume of s129 and SNAP25 within presynaptic 5HTT terminals was observed in DLB overall (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.288, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012), non-depressed DLB (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.313, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016) and DLB with depression (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.316, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.030; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A significant positive correlation was observed between s129 volume and SNAP25 within DAT terminals in DLB cases with depression (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.379, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), but not in DLB cases without depression or controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA significant correlation was observed between VTA neurones and the volume of DAT synapses in sgACC in DLB cases with depression (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.710, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049; Supplementary Fig.\u0026nbsp;2). Furthermore, a significant correlation in DLB cases with depression was observed between VTA neurone number and volume of α-synuclein positive DAT synapses (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.732, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.016; Supplementary Fig.\u0026nbsp;3). No significant correlations were observed between SN neurones and DAT positive synapse volume in sgACC within groups (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMonoaminergic Protein Analysis\u003c/h2\u003e \u003cp\u003eTo determine the impact of DLB on dopaminergic and serotonergic markers we used western and dot blotting. DAT \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.346, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008, tyrosine hydroxylase (TH) \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;8.762, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, dopamine decarboxylase (DDC) \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.660, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018 and dopamine D3 receptor (D3DR) levels \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.913, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.042 were significantly different between the groups in the sgACC (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F). DLB cases overall showed significantly lower DAT (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022), TH (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) and DDC levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024) compared to controls. Significantly lower DAT (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.009), TH (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003), DDC (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.041) and D3DR levels (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049) were observed in DLB cases with depression compared to controls. DLB cases without depression showed significantly lower levels of TH (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.012) compared to controls. No significant difference was observed in dopamine D2 receptors (D2DR) \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.620, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.605 or D4 receptors (D4DR) \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.115, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.109 between groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNo significant changes in 5HTT \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.064, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.978, tryptophan hydroxylase 2 (TPH-2) \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;1.719, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.173, 5HT1\u003csub\u003eA\u003c/sub\u003e receptor \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.258, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.855 or 5HT3\u003csub\u003eB\u003c/sub\u003e receptor \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.156, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.062 was observed between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-L). A significant difference in 5HT2\u003csub\u003eA\u003c/sub\u003e receptor protein levels was observed in the sgACC between groups \u003cem\u003eF\u003c/em\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.858, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019, with significantly lower 5HT2\u003csub\u003eA\u003c/sub\u003e observed in DLB cases overall (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.026) and in DLB cases with depression compared to controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.044).\u003c/p\u003e \u003cp\u003eHeat map analysis showed no clear separation of disease groups based on monoaminergic proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Using linear discriminant analysis to separate cases based on monoaminergic markers to determine the greatest influence on depression showed DAT (Wilks\u0026rsquo; Lambda, 0.535, p\u0026thinsp;=\u0026thinsp;0.0004) and D4DR (Wilks\u0026rsquo; Lambda 0.442, p\u0026thinsp;=\u0026thinsp;0.0002) in combination had greatest predictive values, showing 78% accuracy in re-classifying the original data.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDLB patients are at high risk of developing major depression, with depression having a significant impact on quality of life \u003csup\u003e3\u003c/sup\u003e. Depression in DLB correlates with α-synuclein pathology in late onset MDD, indicating that α-synuclein pathology may be a factor in MDD development in older individuals \u003csup\u003e35\u003c/sup\u003e. The sgACC is an area intimately involved in the aetiology of depression, with structural and functional changes in early onset MDD \u003csup\u003e10, 26\u003c/sup\u003e and a high α-synuclein pathological burden in DLB \u003csup\u003e27\u003c/sup\u003e. Despite high levels of α-synuclein in the sgACC, we found no significant difference between depressed and non-depressed DLB donors for α-synuclein pathology burden suggesting gross pathology within the sgACC is not a significant driver of depressive symptoms. Significant neurone loss within the sgACC in DLB is not apparent, and there is no association with depression. Although the basis for depression in DLB is complex, our results indicate reduced dopaminergic innervation in sgACC with changes in dopaminergic synaptic function and decreased levels of dopaminergic markers contributing. This may warrant clinical trials of D2 dopamine receptor agonists since pramipexole and pergolide show efficacy in reducing depression in patients with PD \u003csup\u003e36, 37\u003c/sup\u003e, as well as symptoms of major depression in patients without PD \u003csup\u003e38, 39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe mesolimbic dopamine pathway from the VTA to cortical and subcortical regions includes the cingulate with additional projections from the dorsal tier of the SN \u003csup\u003e33\u003c/sup\u003e. VTA dopaminergic neurones play a role in reward and stress \u003csup\u003e20\u003c/sup\u003e with dopaminergic dysfunction in the mesolimbic system contributing to anhedonia and loss of motivation in MDD \u003csup\u003e21\u003c/sup\u003e. We identified a decrease in VTA cell number in DLB cases overall, but no significant loss in relation to depression in DLB. Compared to the major loss of neurones in the SN \u003csup\u003e27\u003c/sup\u003e, the VTA shows relative preservation of neurones in PD \u003csup\u003e27, 40\u003c/sup\u003e. Similar to the current findings, severe cell loss and gliosis is associated with the presence of depression in PD \u003csup\u003e41\u003c/sup\u003e, with VTA neuronal density having a significant effect \u003csup\u003e42\u003c/sup\u003e. In PD, PDD, and DLB, higher midbrain α-synuclein burden associates with depressive symptoms \u003csup\u003e27\u003c/sup\u003e suggesting that mesolimbic and mesocortical projections rather than nigrostriatal changes contribute to depression in DLB, similar to PD \u003csup\u003e43\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we observed reduced sgACC dopaminergic fibre density in DLB cases with depression as a potential indicator of the reduced VTA innervation. In PD, motor symptoms appear when around 50% of SN dopaminergic neurones are lost \u003csup\u003e44\u003c/sup\u003e, although striatal innervation at onset of motor symptoms is more severe, showing around 80% terminal loss \u003csup\u003e45\u003c/sup\u003e. Dopaminergic axons and terminals are the main site of pathology, with the neurodegenerative process in PD thought to follow a retrograde pathway originating in the striatal terminals \u003csup\u003e46\u003c/sup\u003e. The enhanced dopaminergic fibre loss seen in this study in DLB cases with depression in the absence of severe (~\u0026thinsp;20%) VTA neuronal loss may represent a similar effect with axon and terminal loss prior to major cell death.\u003c/p\u003e \u003cp\u003eOur STED analysis showed phosphorylated α-synuclein within sgACC dopaminergic synapses and increased phosphorylated α-synuclein in sgACC tissue homogenates. In depressed DLB cases, elevated synaptic α-synuclein corresponded with elevated SNAP25, a finding also seen with serotonergic terminals. One possibility is that pathological α-synuclein sequesters SNAP25 within synapses leading to synaptic dysfunction. Alpha-synuclein modulates synaptic activity through effects on vesicle recycling and release by assisting with SNARE complex formation \u003csup\u003e34, 47\u003c/sup\u003e. The positive correlation identified in the current study may represent a synaptic response to increase activity and maintain normal neurotransmission following reduction in cell numbers \u003csup\u003e48\u003c/sup\u003e. Pathological fibrillar α-synuclein can however, rapidly promote aberrant synaptic activity following neuronal application \u003csup\u003e49\u003c/sup\u003e. In forming aggregates within synapses, fibrillar α-synuclein may cause an effective depletion of functional SNARE proteins including SNAP25, reducing the effective synaptic vesicle pool. Fibrillar and oligomeric α-synuclein also has direct effects on synaptic machinery by depleting SNARE complexes \u003csup\u003e50\u003c/sup\u003e. This may underscore the increase in SNAP25 within α-synuclein containing dopaminergic synapses, with SNAP25 increased, but non-functional due to sequestration in α-synuclein aggregates \u003csup\u003e51\u003c/sup\u003e. The increased volumes of α-synuclein negative synapses in depressed DLB donors may be a compensatory response to reduced function of α-synuclein containing synapses in depressed cases. The combined effects of dopaminergic synapse reduction due to cell loss and reduced synaptic efficiency in remaining synapses may cause an effective depletion of dopamine to the sgACC cortex and contribute to depressive symptoms.\u003c/p\u003e \u003cp\u003eIn this study, we found a loss of dopaminergic proteins in DLB cases with depression compared to controls, including DAT, TH and DDC. Using hierarchical clustering, DAT and DRD4 showed significant associations with depression in DLB. These reductions align with DAT fibre loss in depressed DLB cases and reinforce the general loss of dopaminergic innervation in DLB, but particularly in DLB experiencing depression \u003csup\u003e25, 27\u003c/sup\u003e. Dopaminergic deficits including reduced striatal DAT binding has been observed in depressed patients with anhedonia \u003csup\u003e22\u003c/sup\u003e and additionally in PD and DLB, with DLB cases showing greater DAT loss in caudate compared to PD \u003csup\u003e52\u003c/sup\u003e, with reduced ACC DAT binding in DLB \u003csup\u003e53\u003c/sup\u003e. The use of dopamine agonists in MDD can have significant benefits, and may be beneficial in DLB if suitable treatment regimens are identified \u003csup\u003e54, 55\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur results indicate minimal changes in post-synaptic D2DR in DLB cases. The highest levels of D2DR are found in the striatum, nucleus accumbens and olfactory tubercle, with significant levels in the SN, VTA, amygdala, hippocampus and cortex \u003csup\u003e56\u003c/sup\u003e. In PD, SN neurone loss results in reduced striatal dopamine with increased striatal D2DR as a compensatory mechanism in response to low dopamine \u003csup\u003e57\u003c/sup\u003e. Increased dopamine D2/D3 receptor binding and lower DAT activity has also been shown in MDD, potentially reflecting compensatory changes \u003csup\u003e58, 59\u003c/sup\u003e. This does not appear to occur in DLB however, where reduced striatal and cortical D2DR occurs \u003csup\u003e60, 61\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eD3DR are expressed pre- and postsynaptically and have the highest affinity for dopamine \u003csup\u003e62\u003c/sup\u003e. DRD3 is a clinically relevant treatment target following identification of DRD3 downregulation in MDD \u003csup\u003e63\u003c/sup\u003e. A reduction in DRD3 in the ventral striatum has been observed in PD cases \u003csup\u003e64\u003c/sup\u003e, but with either downregulation \u003csup\u003e65\u003c/sup\u003e or no change \u003csup\u003e60\u003c/sup\u003e in DLB. Our results indicate that reduced DRD3 levels may play a role in depression in DLB, and DRD3 specific dopaminergic agents may be beneficial. Pramipexole and ropinirole improve depressive symptoms in PD and may provide benefit for depression in DLB, although careful monitoring may be needed to prevent unwanted side effects including impulse control disorders and hallucinations \u003csup\u003e54, 66, 67\u003c/sup\u003e. In PD, improved working and episodic memory occurs with dopamine agonists, indicating that dopaminergic agonists may additionally improve cognition \u003csup\u003e68, 69\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur findings show no significant changes in sgACC DRD4 levels in DLB despite an association based on hierarchical clustering. DRD4 shows the lowest levels of expression of dopamine receptors in the brain, with highest densities in anterior limbic and cortical forebrain suggesting cognitive, executive, and motivational regulation \u003csup\u003e70\u003c/sup\u003e. Similar to previous studies indicating that DRD4 are not implicated in the modulation of depressive-like behaviours \u003csup\u003e71\u003c/sup\u003e, our findings suggest that DRD4 may not be a treatment target.\u003c/p\u003e \u003cp\u003eIn contrast to dopaminergic changes, serotonergic involvement in depression in DLB was limited, comparable with similar studies \u003csup\u003e42, 72\u003c/sup\u003e. Whilst the dorsal raphe nucleus (DRN) shows LB pathology in DLB and PD, studies of neurone loss are variable with reduction \u003csup\u003e73\u003c/sup\u003e or no change in DRN neurones in PD or PDD \u003csup\u003e72, 74\u003c/sup\u003e. In one report in DLB, neuronal loss is present within both the DRN and median raphe \u003csup\u003e75\u003c/sup\u003e. In the current study, due to the tissue sampling strategy and differences in DRN projection sites (Ren et al., 2018), DRN neurone numbers were not assessed. We however found no change in sgACC serotonergic fibre density in DLB similar to findings in the amygdala and dorsal prefrontal cortex in LBD donors with and without depression \u003csup\u003e72\u003c/sup\u003e. We did however find α-synuclein associated with serotonergic synapses and a positive correlation between SNAP25 volume and α-synuclein volume. This finding in 5HT synapses may indicate 5HT terminal dysfunction without a change in innervation in the sgACC since we saw no change in fibre density or 5HTT protein levels. Other studies have also showed no association with 5HTT levels with depression or anxiety in PD \u003csup\u003e76\u003c/sup\u003e. Future studies should however determine the effects of α-synuclein pathology in DLB in the brainstem and midbrain serotonergic system using appropriate stereological approaches.\u003c/p\u003e \u003cp\u003eThere has been considerable \u003cem\u003ein vivo\u003c/em\u003e use of 5HTT ligands in PD and to a lesser extent DLB. In drug na\u0026iuml;ve PD, there were no changes in non-apathetic PD but reductions in [(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)C-N,N-dimethyl-2-(-2-amino-4-cyanophenylthio)-benzylamine (DASB)] retention in apathetic patients in the sgACC when compared to non-apathetic PD suggesting reduced serotonergic innervation \u003csup\u003e77\u003c/sup\u003e. Similarly, reduced SERT assessed with DASB is seen in caudate and ACC in early stage PD and throughout the disease course \u003csup\u003e78\u0026ndash;80\u003c/sup\u003e and at post mortem \u003csup\u003e81\u003c/sup\u003e. In depressed PD patients however, increased DASB retention was observed in several cortical areas although more generally there was no change in DASB retention in ACC in MDD \u003csup\u003e82, 83\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e studies in DLB have used the combined DAT and SERT ligand \u003csup\u003e123\u003c/sup\u003eI-N-ω-fluoropropyl-2β-carbomethoxy-3β-(4-iodophenyl) nortropane (\u003csup\u003e123\u003c/sup\u003eI-FP-CIT) where the assumption is that cortical retention only represents 5HT innervation despite DA terminals being present. Studies with \u003csup\u003e123\u003c/sup\u003eI-FP-CIT have shown either reduced cortical SERT \u003csup\u003e84\u0026ndash;86\u003c/sup\u003e, or no cortical changes \u003csup\u003e87\u003c/sup\u003e. No reduction in SERT was seen in the ACC at post mortem in PDD, although brain regions such as caudate showed reductions \u003csup\u003e88\u003c/sup\u003e. Similarly, in DLB compared to control or non-depressed patients, SERT assessed with cyanoimipramine showed preserved cortical binding in depressed DLB donors \u003csup\u003e89, 90\u003c/sup\u003e. Whilst these prior studies have shown variable outcomes, our findings indicate SERT reductions in DLB are mild or absent and contributions to development of depression are limited.\u003c/p\u003e \u003cp\u003eDecreased serotonin 5HT1\u003csub\u003eA\u003c/sub\u003e binding is inidcated in the ACC in MDD patients using PET \u003csup\u003e14\u003c/sup\u003e however, we observed no alteration of 5HT1\u003csub\u003eA\u003c/sub\u003e protein levels in DLB. This contrasts with findings using the 5HT1a ligand 8-Hydroxy-2-Dipropylaminotetralin showing increased binding but altered K\u003csub\u003eD\u003c/sub\u003e in DLB and PD \u003csup\u003e91\u003c/sup\u003e. This may suggest a change in receptor occupancy due to changes in protein conformation but not protein levels. However, our finding of decreased 5HT2\u003csub\u003eA\u003c/sub\u003e protein in sgACC in DLB donors with depression corresponds with reduced 5HT2\u003csub\u003eA\u003c/sub\u003e binding in ACC and DLPFC in MDD \u003csup\u003e15\u003c/sup\u003e and reduced [\u003csup\u003e3\u003c/sup\u003eH]-ketanserin binding in DLB and PDD \u003csup\u003e92, 93\u003c/sup\u003e. These changes in 5HT2\u003csub\u003eA\u003c/sub\u003e but not 5HT1\u003csub\u003eA\u003c/sub\u003e suggests region specific serotonergic receptor changes that may benefit from selective targeting. Pimavanserin, a 5HT2a selective inverse agonist and antagonist, has been used in psychosis treatment in PD \u003csup\u003e94, 95\u003c/sup\u003e. Despite mixed benefits in MDD \u003csup\u003e96, 97\u003c/sup\u003e, an open label study of Pimavanserin for depression in PD in the absence of psychosis showed delayed benefit in an 8-week trial with 60% of patients showing improved mood \u003csup\u003e98\u003c/sup\u003e although longer-term use of Pimavanserin may show reduced efficacy \u003csup\u003e99\u003c/sup\u003e. The possibility of using Pimavanserin in the treatment of depression in may be warranted in certain DLB patients \u003csup\u003e100\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, these results suggest that there is primarily a reduced dopaminergic innervation in the sgACC in DLB, along with reduced levels of dopaminergic markers and receptors, and changes in dopaminergic synaptic function. Careful treatment with selective dopaminergic agonists or positive allosteric modulators may be beneficial in alleviating depressive symptoms in DLB.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests. The views expressed are those of the author(s) and not necessarily those of the NHS, NIHR or the UK Department of Health.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis project is supported by a studentship through the Alzheimer\u0026rsquo;s Society Doctoral Training Centre. Tissue for this study was provided by the Newcastle Brain Tissue Resource, which is funded in part by a grant from the UK Medical Research Council and the Brains for Dementia research, a joint venture between Alzheimer\u0026rsquo;s Society and Alzheimer\u0026rsquo;s Research UK. The research was also partly funded by the National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre based at Newcastle Hospitals NHS Foundation Trust and Newcastle University.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcKeith IG, Dickson DW, Lowe J, Emre M, O'Brien JT, Feldman H \u003cem\u003eet al.\u003c/em\u003e Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium. Neurology 2005; 65(12): 1863\u0026ndash;1872.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcAleese KE, Colloby S, Attems J, Thomas A, Francis PT. Mixed brain pathologies account for most dementia in the Brains for Dementia Research cohort. 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Parkinsonism Relat Disord 2019; 69: 119\u0026ndash;124.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3953937/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3953937/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn addition to the core clinical features of fluctuating cognition, visual hallucinations, and parkinsonism, individuals with dementia with Lewy bodies (DLB) frequently experience chronic and debilitating major depression. Treatment of depression in DLB is hampered by a lack of available effective therapies and standard serotonergic medication for major depressive disorder (MDD) is typically ineffective. Dysfunction of dopaminergic neurotransmission contributing to anhedonia and loss of motivation has been described in MDD. The subgenual anterior cingulate cortex (sgACC) is important in mood regulation and in the symptomatic expression of depression, displaying structural, functional and metabolic abnormalities in MDD. To assess dopaminergic and serotonergic synaptic changes in DLB, post mortem sgACC tissue from DLB donors with and without depression was investigated using high-resolution stimulated emission depletion (STED) microscopy, as well as Western and dot blotting techniques. STED imaging demonstrated the presence of α-synuclein within individual dopaminergic terminals in the sgACC, α-synuclein presence showing a significant positive correlation with increased SNAP25 volumes in depressed DLB cases. A reduction in dopaminergic innervation in the sgACC was observed in DLB cases with depression, along with reduced levels of multiple dopaminergic markers and receptors. Limited alterations were observed in serotonergic markers. Our work demonstrates a role for dopaminergic neurotransmission in the aetiology of depression in DLB. Careful and selective targeting of dopaminergic systems may be a therapeutic option for treatment of depression in DLB.\u003c/p\u003e","manuscriptTitle":"Dopaminergic Changes in the Subgenual Cingulate Cortex in Dementia with Lewy Bodies Associates with Presence of Depression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-18 17:06:02","doi":"10.21203/rs.3.rs-3953937/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"71156a6b-5aca-4255-b76f-e0b8feb7769d","owner":[],"postedDate":"March 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29425754,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":29425755,"name":"Health sciences/Biomarkers/Predictive markers"}],"tags":[],"updatedAt":"2025-03-05T19:50:31+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-18 17:06:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3953937","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3953937","identity":"rs-3953937","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}