The AAV-α-Synuclein Model of Parkinson's Disease: An Update.

Targeted delivery of α-synuclein using AAV vectors has over the two decades since its introduction developed into a versatile tool for modeling different aspects of synucleinopathy, mimicking those seen in Parkinson's disease and related Lewy body disorders. The viral vector approach to disease modeling is attractive in that the expression of α-synuclein, wild-type or mutated, can be confined to defined anatomical structures and targeted to selected cell populations using either cell-type specific promoter constructs or different natural or engineered AAV serotypes. AAV-α-synuclein was initially used to model progressive α-synuclein pathology in nigral dopamine neurons, and, like the standard 6-OHDA model, it has most commonly been applied unilaterally, using the non-injected side as a reference and control. In recent years, however, the AAV-α-synuclein model has become more widely used to induce Parkinson-like synuclein pathology in other relevant neuronal systems, such as the brainstem noradrenergic and serotonergic neurons, the vagal motor neurons, as well as in oligodendrocytes, the prime target relevant to the pathology seen in multiple system atrophy. The purpose of this review is to give an overview of the progress made in the use of the AAV-α-synuclein model over the last two decades and summarize the state-of-the art in the use of the AAV-α-synuclein model for disease modeling in rats and mice.

Misfolding of the neuronal protein α-synuclein is central to the cellular processes that underlie the development of Parkinson's disease and related disorders, such as dementia with Lewy bodies and multiple system atrophy. Targeted delivery of α-synuclein using adeno-associated virus, AAV, has become a standard tool to model the disease process in animals. This AAV-α-synuclein model of Parkinson's disease was introduced two decades ago and over the ensuing decades it has become a widely used standard tool for experimental studies in animals. The usefulness of the AAV-α-synuclein model is largely due to its flexibility and versatility as an experimental tool. In this review the authors summarize the state-of-the art in this field and review the range of applications that has been developed using AAV-α-synuclein alone, in single hit models, or in combinations with other interacting risk factors, in double hit models.

Preclinical quality, safety, and efficacy of a human embryonic stem cell-derived product for the treatment of Parkinson's disease, STEM-PD.

Cell replacement therapies for Parkinson's disease (PD) based on transplantation of pluripotent stem cell-derived dopaminergic neurons are now entering clinical trials. Here, we present quality, safety, and efficacy data supporting the first-in-human STEM-PD phase I/IIa clinical trial along with the trial design. The STEM-PD product was manufactured under GMP and quality tested in vitro and in vivo to meet regulatory requirements. Importantly, no adverse effects were observed upon testing of the product in a 39-week rat GLP safety study for toxicity, tumorigenicity, and biodistribution, and a non-GLP efficacy study confirmed that the transplanted cells mediated full functional recovery in a pre-clinical rat model of PD. We further observed highly comparable efficacy results between two different GMP batches, verifying that the product can be serially manufactured. A fully in vivo-tested batch of STEM-PD is now being used in a clinical trial of 8 patients with moderate PD, initiated in 2022.

Protocol for optical clearing and imaging of fluorescently labeled ex vivo rat brain slices.

Tissue clearing is commonly used for whole-brain imaging but seldom used for brain slices. Here, we present a simple protocol to slice, immunostain, and clear sections of adult rat brains for subsequent high-resolution confocal imaging. The protocol does not require toxic reagents or specialized equipment. We also provide instructions for culturing of rat brain slices free floating on permeable culture inserts, maintained in regular CO2 incubators, and handled only at media change.

A Combined α-Synuclein/Fibril (SynFib) Model of Parkinson-Like Synucleinopathy Targeting the Nigrostriatal Dopamine System.

Injections of pre-formed α-synuclein fibrils (PFFs) or overexpression of α-synuclein using AAV vectors are commonly used as models of Parkinson-like synucleinopathy in rats and mice. In the modified method reviewed here, the "SynFib" model, the PFFs and the AAV vector are administered together unilaterally into the substantia nigra. This approach combines the key features of these two models, i.e., the generation of toxic α-synuclein aggregates and Lewy body-like inclusions, in combination with the increased vulnerability caused by increased cellular levels of α-synuclein. The combined AAV/PFF delivery offers several advantages over the standard PFF model due to the enhanced and accelerated α-synuclein pathology and microglial response induced by the PFF seeds in the presence of an elevated α-synuclein level. Injection of the AAV/PFF mixture into the substantia nigra makes it possible to target a larger proportion of the nigral dopamine neurons and obtain a level of dopamine cell loss (>60%) needed to induce significant impairments in drug-induced and spontaneous motor tests. The SynFib model shares attractive features of the standard 6-OHDA lesion model: a single unilateral stereotaxic intervention; pathology and cell loss developing over a short time span; and the possibility to monitor the degenerative changes using tests of motor behavior.

Single-cell transcriptional and functional analysis of dopaminergic neurons in organoid-like cultures derived from human fetal midbrain.

Significant efforts are ongoing to develop refined differentiation protocols to generate midbrain dopamine (DA) neurons from pluripotent stem cells for application in disease modeling, diagnostics, drug screening and cell-based therapies for Parkinson's disease. An increased understanding of the timing and molecular mechanisms that promote the generation of distinct subtypes of human midbrain DA during development will be essential for guiding future efforts to generate molecularly defined and subtype-specific DA neurons from pluripotent stem cells. Here, we use droplet-based single-cell RNA sequencing to transcriptionally profile the developing human ventral midbrain (VM) when the DA neurons are generated (6-11 weeks post-conception) and their subsequent differentiation into functional mature DA neurons in primary fetal 3D organoid-like cultures. This approach reveals that 3D cultures are superior to monolayer conditions for their ability to generate and maintain mature DA neurons; hence, they have the potential to be used for studying human VM development. These results provide a unique transcriptional profile of the developing human fetal VM and functionally mature human DA neurons that can be used to guide stem cell-based therapies and disease modeling approaches in Parkinson's disease.

Single-cell transcriptomics captures features of human midbrain development and dopamine neuron diversity in brain organoids.

Three-dimensional brain organoids have emerged as a valuable model system for studies of human brain development and pathology. Here we establish a midbrain organoid culture system to study the developmental trajectory from pluripotent stem cells to mature dopamine neurons. Using single cell RNA sequencing, we identify the presence of three molecularly distinct subtypes of human dopamine neurons with high similarity to those in developing and adult human midbrain. However, despite significant advancements in the field, the use of brain organoids can be limited by issues of reproducibility and incomplete maturation which was also observed in this study. We therefore designed bioengineered ventral midbrain organoids supported by recombinant spider-silk microfibers functionalized with full-length human laminin. We show that silk organoids reproduce key molecular aspects of dopamine neurogenesis and reduce inter-organoid variability in terms of cell type composition and dopamine neuron formation.

Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson's Disease.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson's disease (PD) and they provide the option of using the patient's own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. OBJECTIVE: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. METHODS: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. RESULTS: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. CONCLUSION: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

Light sheet fluorescence microscopy of optically cleared brains for studying the glymphatic system.

Fluid transport in the perivascular space by the glia-lymphatic (glymphatic) system is important for the removal of solutes from the brain parenchyma, including peptides such as amyloid-beta which are implicated in the pathogenesis of Alzheimer's disease. The glymphatic system is highly active in the sleep state and under the influence of certain of anaesthetics, while it is suppressed in the awake state and by other anaesthetics. Here we investigated whether light sheet fluorescence microscopy of whole optically cleared murine brains was capable of detecting glymphatic differences in sleep- and awake-mimicking anaesthesia, respectively. Using light-sheet imaging of whole brains, we found anaesthetic-dependent cerebrospinal fluid (CSF) influx differences, including reduced tracer influx along tertiary branches of the middle cerebral artery and reduced influx along dorsal and anterior penetrating arterioles, in the awake-mimicking anaesthesia. This study establishes that light sheet microscopy of optically cleared brains is feasible for quantitative analyses and can provide images of the entire glymphatic system in whole brains.

Impact of α-synuclein pathology on transplanted hESC-derived dopaminergic neurons in a humanized α-synuclein rat model of PD.

Preclinical assessment of the therapeutic potential of dopamine (DA) neuron replacement in Parkinson's disease (PD) has primarily been performed in the 6-hydroxydopamine toxin model. While this is a good model to assess graft function, it does not reflect the pathological features or progressive nature of the disease. In this study, we establish a humanized transplantation model of PD that better recapitulates the main disease features, obtained by coinjection of preformed human α-synuclein (α-syn) fibrils and adeno-associated virus (AAV) expressing human wild-type α-syn unilaterally into the rat substantia nigra (SN). This model gives rise to DA neuron dysfunction and progressive loss of DA neurons from the SN and terminals in the striatum, accompanied by extensive α-syn pathology and a prominent inflammatory response, making it an interesting and relevant model in which to examine long-term function and integrity of transplanted neurons in a PD-like brain. We transplanted DA neurons derived from human embryonic stem cells (hESCs) into the striatum and assessed their survival, growth, and function over 6 to 18 wk. We show that the transplanted cells, even in the presence of ongoing pathology, are capable of innervating the DA-depleted striatum. However, on closer examination of the grafts, we found evidence of α-syn pathology in the form of inclusions of phosphorylated α-syn in a small fraction of the grafted DA neurons, indicating host-to-graft transfer of α-syn pathology, a phenomenon that has previously been observed in PD patients receiving fetal tissue grafts but has not been possible to demonstrate and study in toxin-based animal models.

hESC-Derived Dopaminergic Transplants Integrate into Basal Ganglia Circuitry in a Preclinical Model of Parkinson's Disease.

Cell replacement is currently being explored as a therapeutic approach for neurodegenerative disease. Using stem cells as a source, transplantable progenitors can now be generated under conditions compliant with clinical application in patients. In this study, we elucidate factors controlling target-appropriate innervation and circuitry integration of human embryonic stem cell (hESC)-derived grafts after transplantation to the adult brain. We show that cell-intrinsic factors determine graft-derived axonal innervation, whereas synaptic inputs from host neurons primarily reflect the graft location. Furthermore, we provide evidence that hESC-derived dopaminergic grafts transplanted in a long-term preclinical rat model of Parkinson's disease (PD) receive synaptic input from subtypes of host cortical, striatal, and pallidal neurons that are known to regulate the function of endogenous nigral dopamine neurons. This refined understanding of how graft neurons integrate with host circuitry will be important for the design of clinical stem-cell-based replacement therapies for PD, as well as for other neurodegenerative diseases.

Target-specific forebrain projections and appropriate synaptic inputs of hESC-derived dopamine neurons grafted to the midbrain of parkinsonian rats.

Dopamine (DA) neurons derived from human embryonic stem cells (hESCs) are a promising unlimited source of cells for cell replacement therapy in Parkinson's disease (PD). A number of studies have demonstrated functionality of DA neurons originating from hESCs when grafted to the striatum of rodent and non-human primate models of PD. However, several questions remain in regard to their axonal outgrowth potential and capacity to integrate into host circuitry. Here, ventral midbrain (VM) patterned hESC-derived progenitors were grafted into the midbrain of 6-hydroxydopamine-lesioned rats, and analyzed at 6, 18, and 24 weeks for a time-course evaluation of specificity and extent of graft-derived fiber outgrowth as well as potential for functional recovery. To investigate synaptic integration of the transplanted cells, we used rabies-based monosynaptic tracing to reveal the origin and extent of host presynaptic inputs to grafts at 6 weeks. The results reveal the capacity of grafted neurons to extend axonal projections toward appropriate forebrain target structures progressively over 24 weeks. The timing and extent of graft-derived dopaminergic fibers innervating the dorsolateral striatum matched reduction in amphetamine-induced rotational asymmetry in the animals where recovery could be observed. Monosynaptic tracing demonstrated that grafted cells integrate with host circuitry 6 weeks after transplantation, in a manner that is comparable with endogenous midbrain connectivity. Thus, we demonstrate that VM patterned hESC-derived progenitors grafted to midbrain have the capacity to extensively innervate appropriate forebrain targets, integrate into the host circuitry and that functional recovery can be achieved when grafting fetal or hESC-derived DA neurons to the midbrain.

What factors motivate junior doctors to engage as clinical tutors? A qualitative study.

OBJECTIVES: The study aimed to explore and identify factors motivating junior doctors to engage as long-term clinical tutors in undergraduate medical education. METHODS: In this qualitative study, twenty-seven participants were recruited among junior doctors attending preparatory tutor courses at the Sahlgrenska Academy, University of Gothenburg, and the Primary Healthcare system, West Sweden. They were asked to respond to open-ended questions and write a short account of their needs as clinical tutors for medical students. A qualitative content analysis was performed. RESULTS: A main theme emerged: "Let me develop my skills in a supportive workplace, provide feedback and merits, and I will continue tutoring". Participants described suitable personality as fundamental, and the need to develop professional skills, both as clinical tutors and physicians. Tutor education was an important source of knowledge and stimulation. A workplace environment, supporting learning and the tutor's role, was considered important, including having an adequate time frame. A clear and well-prepared assignment was regarded essential. Junior doctors requested feedback and merits in their work as long-term tutors. Clinical tutorship was considered an optional task. CONCLUSIONS: In this exploratory study, motivating factors of junior doctors' engagement as future long-term tutors were identified. It is important to form a process where junior doctors can build up professional competence as clinical tutors and physicians. To ensure a sustainable tutorship in the future, we suggest that universities and healthcare authorities acknowledge and further study these motivating factors.

Chemogenetic modulation of cholinergic interneurons reveals their regulating role on the direct and indirect output pathways from the striatum.

The intricate balance between dopaminergic and cholinergic neurotransmission in the striatum has been thoroughly difficult to characterize. It was initially described as a seesaw with a competing function of dopamine versus acetylcholine. Recent technical advances however, have brought this view into question suggesting that the two systems work rather in concert with the cholinergic interneurons (ChIs) driving dopamine release. In this study, we have utilized two transgenic Cre-driver rat lines, a choline acetyl transferase ChAT-Cre transgenic rat and a novel double-transgenic tyrosine hydroxylase TH-Cre/ChAT-Cre rat to further elucidate the role of striatal ChIs in normal motor function and in Parkinson's disease. Here we show that selective and reversible activation of ChIs using chemogenetic (DREADD) receptors increases locomotor function in intact rats and potentiate the therapeutic effect of L-DOPA in the rats with lesions of the nigral dopamine system. However, the potentiation of the L-DOPA effect is accompanied by an aggravation of L-DOPA induced dyskinesias (LIDs). These LIDs appear to be driven primarily through the indirect striato-pallidal pathway since the same effect can be induced by the D2 agonist Quinpirole. Taken together, the results highlight the intricate regulation of balance between the two output pathways from the striatum orchestrated by the ChIs.

Modeling Parkinson's disease pathology by combination of fibril seeds and α-synuclein overexpression in the rat brain.

Although a causative role of α-synuclein (α-syn) is well established in Parkinson's disease pathogenesis, available animal models of synucleinopathy do not replicate the full range of cellular and behavioral changes characteristic of the human disease. This study was designed to generate a more faithful model of Parkinson's disease by injecting human α-syn fibril seeds into the rat substantia nigra (SN), in combination with adenoassociated virus (AAV)-mediated overexpression of human α-syn, at levels that, by themselves, are unable to induce acute dopamine (DA) neurodegeneration. We show that the ability of human α-syn fibrils to trigger Lewy-like α-synuclein pathology in the affected DA neurons is dramatically enhanced in the presence of elevated levels of human α-syn. This synucleinopathy was fully developed already 10 days after fibril injection, accompanied by progressive degeneration of dopaminergic neurons in SN, neuritic swelling, reduced striatal DA release, and impaired motor behavior. Moreover, a prominent inflammatory response involving both activation of resident microglia and infiltration of CD4+ and CD8+ T lymphocytes was observed. Hypertrophic microglia were found to enclose or engulf cells and processes containing Lewy-like α-syn aggregates. α-Syn aggregates were also observed inside these cells, suggesting transfer of phosphorylated α-syn from the affected nigral neurons. The nigral pathology triggered by fibrils in combination with AAV-mediated overexpression of α-syn reproduced many of the cardinal features of the human disease. The short time span and the distinct sequence of pathological and degenerative changes make this combined approach attractive as an experimental model for the assessment of neuroprotective and disease-modifying strategies.

DREADD Modulation of Transplanted DA Neurons Reveals a Novel Parkinsonian Dyskinesia Mechanism Mediated by the Serotonin 5-HT6 Receptor.

Transplantation of DA neurons is actively pursued as a restorative therapy in Parkinson's disease (PD). Pioneering clinical trials using transplants of fetal DA neuroblasts have given promising results, although a number of patients have developed graft-induced dyskinesias (GIDs), and the mechanism underlying this troublesome side effect is still unknown. Here we have used a new model where the activity of the transplanted DA neurons can be selectively modulated using a bimodal chemogenetic (DREADD) approach, allowing either enhancement or reduction of the therapeutic effect. We show that exclusive activation of a cAMP-linked (Gs-coupled) DREADD or serotonin 5-HT6 receptor, located on the grafted DA neurons, is sufficient to induce GIDs. These findings establish a mechanistic link between the 5-HT6 receptor, intracellular cAMP, and GIDs in transplanted PD patients. This effect is thought to be mediated through counteraction of the D2 autoreceptor feedback inhibition, resulting in a dysplastic DA release from the transplant.

Extensive graft-derived dopaminergic innervation is maintained 24 years after transplantation in the degenerating parkinsonian brain.

Clinical trials using cells derived from embryonic ventral mesencephalon have shown that transplanted dopaminergic neurons can survive and function in the long term, as demonstrated by in vivo brain imaging using (18)F-fluorodopa and (11)C-raclopride positron emission tomography. Here we report the postmortem analysis of a patient with Parkinson's disease who 24 y earlier underwent unilateral transplantation of embryonic dopaminergic neurons in the putamen and subsequently exhibited major motor improvement and recovery of striatal dopaminergic function. Histopathological analysis showed that a dense, near-normal graft-derived dopaminergic reinnervation of the putamen can be maintained for a quarter of a century despite severe host brain pathology and with no evidence of immune response. In addition, ubiquitin- and α-synuclein-positive inclusions were seen, some with the appearance of typical Lewy bodies, in 11-12% of the grafted dopaminergic neurons, reflecting the spread of pathology from the host brain to the transplants. Because the clinical benefits induced by transplantation in this patient were gradually lost after 14 y posttransplantation, our findings provide the first reported evidence, to our knowledge, that even a viable dopaminergic graft giving rise to extensive striatal reinnervation may lose its efficacy if widespread degenerative changes develop in the host brain.

α-Synuclein induced toxicity in brain stem serotonin neurons mediated by an AAV vector driven by the tryptophan hydroxylase promoter.

We studied the impact of α-synuclein overexpression in brainstem serotonin neurons using a novel vector construct where the expression of human wildtype α-synuclein is driven by the tryptophan hydroxylase promoter, allowing expression of α-synuclein at elevated levels, and with high selectivity, in serotonergic neurons. α-Synuclein induced degenerative changes in axons and dendrites, displaying a distorted appearance, suggesting accumulation and aggregation of α-synuclein as a result of impaired axonal transport, accompanied by a 40% loss of terminals, as assessed in the hippocampus. Tissue levels of serotonin and its major metabolite 5-HIAA remained largely unaltered, and the performance of the α-synuclein overexpressing rats in tests of spatial learning (water maze), anxiety related behavior (elevated plus maze) and depressive-like behavior (forced swim test) was not different from control, suggesting that the impact of the developing axonal pathology on serotonin neurotransmission was relatively mild. Overexpression of α-synuclein in the raphe nuclei, combined with overexpression in basal forebrain cholinergic neurons, resulted in more pronounced axonal pathology and significant impairment in the elevated plus maze. We conclude that α-synuclein pathology in serotonergic or cholinergic neurons alone is not sufficient to impair non-motor behaviors, but that it is their simultaneous involvement that determines severity of such symptoms.

Nurr1 and Retinoid X Receptor Ligands Stimulate Ret Signaling in Dopamine Neurons and Can Alleviate α-Synuclein Disrupted Gene Expression.

α-synuclein, a protein enriched in Lewy bodies and highly implicated in neurotoxicity in Parkinson's disease, is distributed both at nerve terminals and in the cell nucleus. Here we show that a nuclear derivative of α-synuclein induces more pronounced changes at the gene expression level in mouse primary dopamine (DA) neurons compared to a derivative that is excluded from the nucleus. Moreover, by RNA sequencing we analyzed the extent of genome-wide effects on gene expression resulting from expression of human α-synuclein in primary mouse DA neurons. The results implicated the transcription factor Nurr1 as a key dysregulated target of α-synuclein toxicity. Forced Nurr1 expression restored the expression of hundreds of dysregulated genes in primary DA neurons expressing α-synuclein, and therefore prompted us to test the possibility that Nurr1 can be pharmacologically targeted by bexarotene, a ligand for the retinoid X receptor that forms heterodimers with Nurr1. Although our data demonstrated that bexarotene was ineffective in neuroprotection in rats in vivo, the results revealed that bexarotene has the capacity to coregulate subsets of Nurr1 target genes including the receptor tyrosine kinase subunit Ret. Moreover, bexarotene was able to restore dysfunctional Ret-dependent neurotrophic signaling in α-synuclein-overexpressing mouse DA neurons. These data highlight the role of the Nurr1-Ret signaling pathway as a target of α-synuclein toxicity and suggest that retinoid X receptor ligands with appropriate pharmacological properties could have therapeutic potential in Parkinson's disease. SIGNIFICANCE STATEMENT: How α-synuclein, a protein enriched in Lewy bodies in Parkinson's disease, is causing neuropathology in dopamine neurons remains unclear. This study elucidated how α-synuclein is influencing gene expression and how Nurr1, a transcription factor known to protect dopamine neurons against α-synuclein toxicity, can counteract these effects. Moreover, given the protective role of Nurr1, this study also investigated how Nurr1 could be pharmacologically targeted via bexarotene, a ligand of Nurr1's heterodimerization partner retinoid X receptor (RXR). The results showed that RXR ligands could increase neurotrophic signaling, but provided a mixed picture of its potential in a Parkinson's disease rat model in vivo. However, this study clearly emphasized Nurr1's neuroprotective role and indicated that other RXR ligands could have therapeutic potential in Parkinson's disease.

Student-centred GP ambassadors: Perceptions of experienced clinical tutors in general practice undergraduate training.

OBJECTIVE: To explore experienced general practitioner (GP) tutor perceptions of a skilled GP tutor of medical students. DESIGN: Interview study based on focus groups. SETTING: Twenty GPs experienced in tutoring medical students at primary health care centres in two Swedish regions were interviewed. METHOD: Four focus-group interviews were analysed using qualitative content analysis. SUBJECTS: Twenty GP tutors, median age 50, specifically selected according to age, gender, and location participated in two focus groups in Gothenburg and Malmö, respectively. MAIN OUTCOME MEASURES: Meaning units in the texts were extracted, coded and condensed into categories and themes. RESULTS: Three main themes emerged: "Professional as GP and ambassador to general practice", "Committed and student-centred educator", and "Coordinator of the learning environment". CONCLUSION: Experienced GP tutors describe their skills as a clinical tutor as complex and diversified. A strong professional identity within general practice is vital and GP tutors describe themselves as ambassadors to general practice, essential to the process of recruiting a new generation of general practitioners. Leaders of clinical education and health care planners must understand the complexity in a clinical tutor's assignment and provide adequate support, time, and resources in order to facilitate a sustainable tutorship and a good learning environment, which could also improve the necessary recruitment of future GPs.