Unravelling neurotransmitters impairment in primary progressive aphasias

Abstract Primary progressive aphasias (PPAs) are a group of neurodegenerative diseases mainly characterized by language impairment, and with variably presence of dysexecutive syndrome, behavioural disturbances and parkinsonism. Detailed knowledge of neurotransmitters impairment and its association with clinical features hold the potential to develop new tailored therapeutic approaches. In the present study, we applied JuSpace toolbox, which allowed for cross‐modal correlation of magnetic resonance imaging (MRI)‐based measures with nuclear imaging derived estimates covering various neurotransmitter systems including dopaminergic, serotonergic, noradrenergic, GABAergic and glutamatergic neurotransmission. We included 103 PPA patients and 80 age‐matched healthy controls (HC). We tested if the spatial patterns of grey matter volume (GMV) alterations in PPA patients (relative to HC) are correlated with specific neurotransmitter systems. As compared to HC, voxel‐based brain changes in PPA were significantly associated with spatial distribution of serotonin, dopamine, and glutamatergic pathways (p < .05, False Discovery Rate corrected‐corrected). Disease severity was negatively correlated with the strength of GMV colocalization of D1 receptors (p = .035) and serotonin transporter (p = .020). Moreover, we observed a significant negative correlation between positive behavioural symptoms, as measured with Frontal Behavioural Inventory, and GMV colocalization of D1 receptors (p = .007) and serotonin transporter (p < .001). This pilot study suggests that JuSpace is a helpful tool to indirectly assess neurotransmitter deficits in neurodegenerative dementias and may provide novel insight into disease mechanisms and associated clinical features.

Moreover, we observed a significant negative correlation between positive behavioural symptoms, as measured with Frontal Behavioural Inventory, and GMV colocalization of D1 receptors (p = .007) and serotonin transporter (p < .001). This pilot study suggests that JuSpace is a helpful tool to indirectly assess neurotransmitter deficits in neurodegenerative dementias and may provide novel insight into disease mechanisms and associated clinical features.

K E Y W O R D S
behavioural disturbances, magnetic resonance imaging, neurotransmitters, positron emission tomography, primary progressive aphasia 1 | INTRODUCTION Primary progressive aphasias (PPAs) are a group of neurodegenerative diseases presenting with insidious and relentless language impairment (Gorno-Tempini et al., 2011;Rosen et al., 2006;Van Langenhove et al., 2016). Two main PPA variants have been described within the spectrum of frontotemporal lobar degeneration: the nonfluent/ agrammatic variant (avPPA), presenting with slow, effortful, hesitant and distorted speech, and the semantic variant (svPPA), which begins as difficulty finding words, particularly nouns, and single words comprehension deficits (Gorno-Tempini et al., 2011;Marshall et al., 2018).
Up to now, PPA interventions rely mainly on speech training (Pagnoni et al., 2021), and on promising approaches with noninvasive brain stimulation techniques such as transcranial Direct Current Stimulation (tDCS) or repetitive Transcranial Magnetic Stimulation Cotelli et al., 2014;Pytel et al., 2021;Tsapkini et al., 2018) to counteract language deficits. However, it remains important to advance symptomatic treatment, to reduce disease burden and improve patients' and carers' quality of life (Murley & Rowe, 2018).
In this view, restoring neurotransmitters deficits hold the potential to improve associated behavioural, cognitive and motor symptoms in PPAs, as a number of studies, mainly performed in autopsy case series, have reported consistent impairment of dopaminergic, serotoninergic, GABAergic and glutamatergic pathways (Murley & Rowe, 2018 Recent advancements in positron emission tomography (PET) or single photon computed emission tomography (SPECT) tracer development resulted in a variety of novel tracers that can reliably measure the availability of specific receptors. In line with that, Dukart and colleagues have shown that drug-induced spatial alteration patterns in resting state functional activity as measured using magnetic resonance imaging (rsfMRI) are associated with the distribution of specific receptors systems targeted by respective compounds (Dukart et al., 2018).

Based on this approach, Dukart et al have recently developed
JuSpace, a toolbox aimed at testing the associations between MRIbased measures and a list of included PET and SPECT maps covering various neurotransmitter systems (Dukart et al., 2021). More in detail, JuSpace creates a spatial pattern of brain alterations based on MRI measures, comparing two different groups (e.g. patients versus healthy controls). After that, it performs a correlation between these alterations and each receptor/transporter map included in the toolbox. JuSpace therefore aims to assess if the spatial patterns of brain changes observed in the disease of interest are related to the distribution of specific neurotransmitters systems, as derived from independent healthy volunteer populations (Dukart et al., 2021).
These premises prompted the present study, aimed at applying JuSpace tool in a large sample of subjects with PPA, with the aim to evaluate the pattern of neurotransmitters deficits in avPPA and svPPA, and the correlation between neurotransmitter deficits and clinical symptoms. Story, Trail Making test A and B, as previously published (Borroni et al., 2010;Premi et al., 2016). Behavioural disturbances were assessed with Frontal Behavioural Inventory (Borroni et al., 2010;Premi et al., 2016). Basic activities of daily living (BADL) and instrumental activities of daily living (IADL) were also assessed, and disease severity was measured by CDR Dementia Staging Instrument plus behaviour and language domains from the National Alzheimer's Coordinating Center and Frontotemporal lobar degeneration modules-sum of boxes (CDR plus NACC FTLD-SOB) (Miyagawa et al., 2020). uni-jena.de/cat) for SPM12 (SPM12 v.7219) (www.fil.ion.ucl.ac.uk/ spm/software/spm12) running on MATLAB R2019b (the MathWorks, Inc., Natick, MA, USA). The VBM pipeline consists of several stages (tissue segmentation, spatial normalization to a standard Montreal National Institute [MNI] template, modulation and smoothing), as previously described (Kurth et al., 2015). CAT12 potentially provides more robust and accurate performances compared to other VBM pipelines (Farokhian et al., 2017). The normalized and modulated grey matter images were then smoothed with 10-mm full width at halfmaximum Gaussian kernel.
To test for group differences (avPPA vs. HC and svPPA vs. HC) in grey matter volume (GMV) a general linear model using SPM12 was implemented, considering age, gender and scanner as nuisance variables. The statistical threshold was set to p < .05 corrected for multiple comparisons (whole-brain family-wise error-FWE).

| Spatial correlation with neurotransmitter density maps
We used the JuSpace toolbox (version 1.4) to test if the spatial patterns of GMV alterations in avPPA or svPPA patients (relative to HC) are correlated with specific neurotransmitter systems (Dukart et al., 2021). Confounding effects of age, gender and scanner type were regressed out from all images prior to these analyses (Dukart et al., 2021). We tested if the spatial structure of GMV maps in patients relative to HC was similar to the distribution of average nuclear imaging derived neurotransmitter maps, derived from independent healthy volunteer populations included in the toolbox. We considered the 5-hydroxytryptamine 1a (5-HT1a) receptor, the 5-HT2a receptor, the serotonin transporter SERT, the D1 receptor, the D2 receptor, the dopamine transporter (DAT), the FluoroDOPA, the GABAa receptors, the vesicular acetylcholine transporter (VAChT), the metabotropic glutamate receptor type 5 (mGLUR5), and the noradrenaline transporter (NAT), to assess serotonin, dopamine, GABAergic and glutamatergic systems. Each map included in Juspace toolbox was available from the literature, as reported in Table S1. Spearman correlation coefficients (Fisher's Z transformed, performed to ensure a normal distribution of the obtained correlation coefficients and to allow subsequent parametric testing) were calculated between these Z-transformed GMV maps and the spatial distribution of the respective neurotransmitter maps. A negative correlation (and resulting negative Fisher's Z) means that GMV decline in patients is strongest in regions with an initial (healthy) high availability of the studied receptor (as derived from healthy independent populations), pointing to an increased disease vulnerability of the respective regions.
Exact permutation-based p-values as implemented in JuSpace (10,000 permutations randomly assigning group labels using orthogonal permutations) were computed to test if the observed correlation coefficients across patients deviate from a null distribution.

| Statistical analysis
Comparisons of demographic and clinical characteristics were performed by the Student's t-test for continuous variables and the χ 2 test for categorical variables.
Spearman correlation was used to assess the relationship between each neurotransmitter output obtained with Juspace (i.e., the GMV-neurotransmitters correlation, Fisher's Z transformed) and clinical or behavioural data. To assess the contributions of neurotransmitters output to behavioural disturbances, we computed binary logistic regression models considering GMV-neurotransmitters correlations as independent variables throughout different behavioural groups. Statistical significance was set at p < .05, corrected for multiple comparisons (False Discovery Rate-FDR) (SPSS Statistics 22.0, Chicago, IL, USA).

| Data availability
All study data, including raw and analysed data, and materials will be available from the corresponding author, B.B., upon reasonable request. The software applied is publicly available at https://github. com/juryxy/JuSpace.

| Neurotransmitters deficits in avPPA and svPPA
As compared to HC, voxel-based brain changes in avPPA were significantly associated with spatial distribution of 5-HT1a receptors For both groups, the negative correlation coefficients indicate GMV reduction in patients as compared to HC in areas with high neurotransmitters density.

| Neurotransmitter impairment and clinical symptoms in PPA
Next, we assessed the relationship between GMV-neurotransmitters correlation coefficients and clinical or behavioural data in PPA group.
We considered only GMV-neurotransmitters correlation coefficients significantly impaired in PPA and we excluded those highly correlated to each other (Spearman correlations coefficients > .80), namely DAT and FluoroDOPA.
Disease severity as measured by CDR plus NACC SOB was negatively correlated with the strength of GMV colocalization of D1 receptors (r = À.211, p = .035) and SERT (r = À.233, p = .020), with greater disease severity being associated with lower GMVneurotransmitters correlation coefficients (see Figure 3).
Similarly, we observed a significant negative correlation between FBI-B, that is, positive behavioural symptoms, and GMV colocalization of D1 receptors (r = À.277, p = .007) and SERT (r = À.358, p < .001), with more severe behavioural symptoms being associated with lower GMV-neurotransmitters correlation coefficients; no significant correlation was detected between FBI-A, that is, negative behavioural symptoms, and any GMV co-localization of neurotransmitters in PPA.
F I G U R E 1 Voxel-wise analyses in avPPA and in svPPA as compared to HC.

| DISCUSSION
The knowledge of neurotransmitters impairment in PPAs and its association with clinical features may be helpful for future tailored therapeutic approaches (Johnson et al., 2010;Kertesz et al., 2008).
Restoring these deficits, individually or in combination, has the potential to improve clinical symptoms and quality of life in both patients and their carriers.
In the present work, we assessed if the spatial patterns of grey matter atrophy observed in different subtypes of PPA are related to the distribution of specific neurotransmitters systems as derived from independent healthy volunteer populations (Dukart et al., 2021).
These data have been obtained by JuSpace toolbox, an integrated system for the comparison of PET and SPECT derived neurotransmitter maps with other imaging modalities such as MRI data (Dukart et al., 2021).
We found that grey matter volume alterations in patients with PPA significantly co-localized with dopaminergic, serotoninergic and glutamatergic circuits. These findings corroborated previous literature autopsy data (Murley & Rowe, 2018), with a new in vivo imaging approach, and also allowed us to infer on correlations between neurotransmitter pathways and clinical presentation in a relative large sample of patients.
Indeed, we also demonstrated that disease severity was associated with progressive worsening of dopamine and serotonin circuits, and more interestingly, impulsivity and binge eating disturbances were strongly associated with grey matter volume co-localization to these neurotransmitter systems.
Indeed, these findings suggest that PPA may be associated with increased vulnerability of dopamine and serotonin systems to atrophy inducing disease pathology. In this view, targeting selective neurotransmitters circuits may be considered in future clinical trials to counteract disease course. Moreover, the major idea behind this approach is that pharmacological manipulation of specific neurotransmitters in PPA may be beneficial only considering associated clinical and behavioural features, thus reducing the cost and health burden of the disease. The core feature of PPA is the language deficit, but behavioural disturbances are frequently detected over disease course (Banks & Weintraub, 2009;de la Sablonnière et al., 2021;Fatemi et al., 2011;G omez-Tortosa et al., 2016;Van Langenhove et al., 2016).
Most studies evaluating pharmacological approaches in frontotemporal lobar degeneration have not reported clear-cut results (Panza et al., 2020). Herein, we suggested that dopamine and serotonin pathways, and in particular D1 receptors and SERT, may be key in the onset of impulsivity and binge eating. Indeed, our findings confirm and extend previous literature data, which have already linked these behavioural disturbances to both dopamine and serotonin abnormalities (da Cunha-Bang & Knudsen, 2021; Li et al., 2021;Majuri et al., 2017). Taken together, the present results, even though preliminary, provide a framework for additional studies to examine symptom-specific pharmacological treatment approaches.
As compared to data obtained in previous neurophysiological studies (Benussi et al., 2017;Benussi, Grassi, Palluzzi, et al., 2020), we failed to confirm a colocalization of grey matter alteration and the GABAergic system, and we found significant glutamatergic impairment only in avPPA, even though we analysed metabotropic receptors and we did not consider NMDA glutamatergic receptors (Benussi et al., 2017;Benussi, Grassi, Palluzzi, et al., 2020).
We acknowledge that this study entails some limitations. First, future implemented neurotransmitters maps in JuSpace may further refine the present findings. Moreover, the maps available have been recently obtained, and present some limitations that might to be addressed, for example, the variability in the number of HC cases in each map and receptor density assessment is not necessarily related to neurotransmitter density. In spite of this, JuSpace appears a promising tool to explore in vivo neurotransmitter impairment (Dukart et al., 2021) (Premi et al., 2014). Third, considering frontotemporal lobar degeneration phenotypes with prominent behavioural disturbances, such as behavioural variant frontotemporal dementia, may be of further interest.

| CONCLUSIONS
This pilot study suggests that JuSpace is a helpful tool to indirectly assess neurotransmitter deficits in neurodegenerative dementias and may provide novel insight into disease mechanisms and associated clinical features. This approach may be useful to design and test new targets for pharmacological intervention trials.

ACKNOWLEDGEMENT
The authors thank patients and their families for entering the study.

FUNDING INFORMATION
This study was supported by grants from Italian Ministry of Health (ACROSS Study, GR-2018-12365105 and Ricerca Corrente).

CONFLICT OF INTEREST
No competing interests were disclosed.

DATA AVAILABILITY STATEMENT
All study data, including raw and analysed data, and materials will be available from the corresponding author, B.B., upon reasonable request. The software applied is publicly available at https://github.

ETHICS STATEMENT
The project has been approved by the local ethics committee of Brescia Hospital and of the IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Italy. The procedures used in this study adhere to the tenets of the Declaration of Helsinki.

INFORMED CONSENT
Each patient signed consent form to participate to the study, and freely given, informed consent to participate in the study was obtained from participants.