This work analyzes the vector dose and exposure time required to induce neuroinflammation, neurodegeneration, and motor impairment in this preclinical model of Parkinson's disease. These vectors encoding the human α-synuclein are delivered into the substantia nigra to recapitulate the synuclein pathology associated with Parkinson's disease.
Parkinson's disease is a neurodegenerative disorder that involves the death of the dopaminergic neurons of the nigrostriatal pathway and, consequently, the progressive loss of control of voluntary movements. This neurodegenerative process is triggered by the deposition of protein aggregates in the brain, which are mainly constituted of α-synuclein. Several studies have indicated that neuroinflammation is required to develop the neurodegeneration associated with Parkinson's disease. Notably, the neuroinflammatory process involves microglial activation as well as the infiltration of peripheral T cells into the substantia nigra (SN). This work analyzes a mouse model of Parkinson's disease that recapitulates microglial activation, T-cell infiltration into the SN, the neurodegeneration of nigral dopaminergic neurons, and motor impairment. This mouse model of Parkinson's disease is induced by the stereotaxic delivery of adeno-associated viral vectors encoding the human wild-type α-synuclein (AAV-hαSyn) into the SN. The correct delivery of viral vectors into the SN was confirmed using control vectors encoding green fluorescent protein (GFP). Afterward, how the dose of AAV-hαSyn administered in the SN affected the extent of hαSyn expression, the loss of nigral dopaminergic neurons, and motor impairment were evaluated. Moreover, the dynamics of hαSyn expression, microglial activation, and T-cell infiltration were determined throughout the time course of disease development. Thus, this study provides critical time points that may be useful for targeting synuclein pathology and neuroinflammation in this preclinical model of Parkinson's disease.
After Alzheimer's disease, Parkinson's disease is the second most prevalent neurodegenerative disease worldwide. The primary neurons affected in Parkinson's disease are those of the nigrostriatal pathway, which produce dopamine and control voluntary movement. As a consequence, the most characteristic symptom associated with this disorder is motor impairment. This pathology also involves the deposition of protein aggregates in the brain, which are composed mainly of α-synuclein (αSyn)1, a cytosolic protein associated to presynaptic terminals. Evidence has shown that the generation of pathogenic inclusions of αSyn is triggered by misfolding or by some post-translational modifications of this protein2.
Notably, a close relationship has been established between αSyn pathology and the loss of dopaminergic neurons of the nigrostriatal pathway in human Parkinson's disease and animal models3,4. Understanding how αSyn aggregates are generated and how they induce neuronal death represents a significant challenge in the field. A growing group of studies has shown that, by increasing oxidative stress, mitochondrial dysfunction is one of the leading causes for the generation of αSyn aggregates2. Indeed, several genes associated with Parkinson's disease risk encode proteins involved in mitochondrial function, morphology, and dynamics5,6. In addition, lysosomal dysfunction, which results in the accumulation of dysfunctional mitochondria and misfolded αSyn constitutes another major event promoting the generation of αSyn aggregates7.
Emerging evidence has indicated that, once αSyn aggregates are deposited in the brain, these pathogenic proteins stimulate toll-like receptors (TLRs) on the microglia, thus triggering microglial activation and an initial inflammatory environment in the substantia nigra (SN)8,9. Furthermore, the evidence indicates that αSyn aggregates are captured and presented by antigen-presenting cells to T cells, inducing an adaptive immune response specific to αSyn10,11. These αSyn-specific T cells subsequently infiltrate the brain and are restimulated by activated microglia, thus promoting the secretion of neurotoxic factors that evoke neuronal death9,10. Interestingly, several lines of evidence have suggested that αSyn aggregates are generated first in the enteric nervous system and then transported through the vagus nerve to the brain stem12.
Several animal models of Parkinson's disease have been used for many years, including those induced by the administration of neurotoxic substances (i.e., 6-hydroxydopamine, paraquat, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and those involving genetic conditions (i.e., mutant α-synuclein, mutant leucine-rich repeat kinase 2)13. Despite models involving neurotoxin-induced neurodegeneration replicating some aspects of Parkinson's disease, none of them recapitulate all the essential aspects of the disease or are not progressive13. On the other hand, although genetic mouse models involving the expression of mutant versions of leucine-rich repeat kinase 2, mutant versions of α-synuclein, or the overexpression of human wild-type α-synuclein result in motor impairment and, in some cases, also the development of synucleinopathy, they do not reproduce prominent neurodegeneration of the nigral dopaminergic neurons, which is an essential aspect of Parkinson's disease13,14. A third kind of animal model of neurodegeneration has managed to meet most of the essential aspects of Parkinson's disease, the stereotaxic delivery of adeno-associated viral vectors (AAVs) encoding the human α-synuclein (AAV-hαSyn)14,15. Importantly, AAVs allow the transduction of neurons with high efficacy and in the long term in the adult brain of mammals. Furthermore, the stereotaxic delivery of AAV-hαSyn in the SN has been shown to reproduce many of the essential aspects of the disease, including αSyn pathology, microglial activation, neurodegeneration, and motor impairment16,17,18,19,20. This study presents an analysis of how the dose of viral vector and the time after viral vector delivery affects the extent of hαSyn expression, neurodegeneration, and neuroinflammation in the nigrostriatal pathway, as well as the degree of motor impairment in the mouse model of unilateral stereotaxic delivery of hαSyn in the SN.
All studies were carried out under the 8th edition of the Guide for the Care and Use of Laboratory Animals. Experimental protocols were approved by the IACUC at the Fundación Ciencia & Vida (Science for Life Foundation), including those involving anesthesia, pain, distress, and euthanasia (Permit number P-035/2022).
1. The stereotaxic surgery
2. Determination of motor performance using the beam test
3. Tissue processing
4. Immunohistochemical analysis to quantify dopaminergic neurons and microgliosis (approximately 2 days)
Target antigen | Coupled to | Clonality | Host specie | Specie reactivity* | Dilution** |
Tyrosine Hydroxylase | N/A | Polyclonal | Rabbit | Mouse, Rat, Human | 1/200 – 1/1000 |
Iba1 | N/A | Monoclonal | Rabbit | Mouse, Rat, Human | 1/1000 |
alpha-Synuclein | N/A | Monoclonal | Rabbit | Human | 1/150 |
CD4 | N/A | Monoclonal | Rat | Mouse | 1/250 |
IgG (H+L) | Biotinilated | Polyclonal | Goat | Rabbit | 1/500 |
IgG (H+L) | AlexaFluor 546 | Polyclonal | Goat | Rabbit | 1/500 |
IgG (H+L) | AlexaFluor 647 | Polyclonal | Goat | Rabbit | 1/500 |
IgG (H+L) | AlexaFluor 546 | Polyclonal | Goat | Rat | 1/500 |
Table 1: Antibody dilutions. N/A, not applicable. *, It is only specified if there are reactivities with mouse, rat, and human, irrespective of the reactivity with other species. **, A single dilution or a dilution range is specified.
5. Immunofluorescence analysis to evaluate T cell infiltration in the nigrostriatal pathway (approximately 2 days)
Chanel Name | Cube | Emission Wavelenght | Lookup Table name | Exposure time | Gain | Resolution XY | Resolution Z |
Channel 1 | Y5 | 700nm | Grey | 1,011.727 ms | high well capacity | 2.237 um | 24.444 um |
Channel 2 | GFP | 525nm | Green | 326.851 ms | high well capacity | 2.237 um | 24.444 um |
Channel 3 | TXR | 630nm | Red | 406.344 ms | high well capacity | 2.237 um | 24.444 um |
Channel 4 | Dapi | 460nm | Blue | 91.501 ms | high well capacity | 2.237 um | 24.444 um |
Table 2: Confocal microscope settings used for the acquisition of images from immunofluorescence analysis.
6. Statistical analysis
Validating the correct delivery of AAV vectors in the dopaminergic neurons of the nigrostriatal pathway
To study the processes of neuroinflammation, neurodegeneration, and motor impairment promoted by synuclein pathology, a mouse model of Parkinson's disease induced by the unilateral stereotaxic delivery of AAV encoding hαSyn in the SN16,17,30,31 was used (see the experimental design in Supplementary Figure 1). To validate the correct delivery of AAV vectors in the dopaminergic neurons of the nigrostriatal pathway, AAV encoding GFP (AAV-GFP) was injected in the SN, and 12 weeks later, GFP fluorescence and tyrosine hydroxylase (TH) immunoreactivity were analyzed in the SN and striatum by immunofluorescence. The GFP-associated fluorescence was observed exclusively on the ipsilateral side, and there was significant colocalization with TH immunoreactivity in both the SN and striatum, indicating the correct delivery of AAV vectors in the dopaminergic neurons of the nigrostriatal pathway (Figure 1).
Figure 1: Analysis of the delivery of AAV-GFP in the nigrostriatal pathway. Mice received AAV-GFP (1 x 1010 vg/mouse) and 12 weeks later were sacrificed, and TH was immunostained in (A) the SN (scale bars are 118 µm) and (B) the striatum (scale bars are 100 µm). TH- and GFP-associated fluorescence were analyzed by epifluorescence microscopy. Nuclei were stained with DAPI. Representative images of merged or single staining of TH (red), GFP (green), and DAPI (blue) are shown. Please click here to view a larger version of this figure.
Setting up the dose of viral vector administered to induce neurodegeneration and motor impairment in the mouse model of Parkinson's disease induced by AAV-hαSyn
To test the dose of AAV-hαSyn required to induce a significant overexpression of hαSyn that promotes neurodegeneration of the nigral dopaminergic neurons, different doses (1 x 108 viral genomes [vg]/mouse, 1 x 109 vg/mouse, or 1 x 1010 vg/mouse) of AAV-hαSyn were injected, and 12 weeks later, hαSyn immunoreactivity and the extent of TH immunoreactivity were evaluated in the nigrostriatal pathway. Although hαSyn immunoreactivity was evident with all doses tested in the SN (Figure 2), only mice receiving 1 x 1010 vg/mouse presented evident hαSyn immunoreactivity in the striatum (Figure 3). Moreover, mice receiving 1 x 1010 vg/mouse of AAV-hαSyn displayed a significant loss of dopaminergic neurons in the SN (Figure 4A,B). Although mice receiving 1 x 1010 vg/mouse of AAV-GFP displayed a low degree (~20%) of neuronal loss (Figure 4A,B), mice receiving the same dose of AAV-hαSyn presented a significantly higher degree of neurodegeneration of nigral dopaminergic neurons (Figure 4C). Accordingly, further experiments were performed using 1 x 1010 vg/mouse of AAV-hαSyn. In addition, the extent of motor impairment was determined in mice receiving different doses of AAV-hαSyn by using the beam test (Figure 5A), as described before25. A significant reduction in motor performance was detected exclusively with 1 x 1010 vg/mouse of AAV-hαSyn in the beam test both when comparing the number of errors made by the right and the left pads (Figure 5B) and when comparing the total number of errors of mice receiving AAV-hαSyn compared with mice receiving the control vector AAV-GFP (Figure 5C). Accordingly, further experiments were performed using 1 x 1010 vg/mouse of AAV-hαSyn.
Figure 2: Analysis of human α-synuclein expression in the SN of mice treated with different doses of AAV-hαSyn. Mice received AAV-hαSyn at (A) 1 x 1010 vg/mouse, (B) 1 x 109 vg/mouse, or (C) 1 x 108 vg/mouse and 12 weeks later were sacrificed, and the hαSyn expression was analyzed by immunofluorescence in the SN using epifluorescence microscopy. Nuclei were stained with DAPI. Representative images of merged or single staining of hαSyn (red) or DAPI (blue) are shown. Scale bars are 118 µm. Please click here to view a larger version of this figure.
Figure 3: Analysis of human α-synuclein expression in the striatum of mice treated with different doses of AAV-hαSyn. Mice received AAV-hαSyn at (A) 1 x 1010 vg/mouse, (B) 1 x 109 vg/mouse, or (C) 1 x 108 vg/mouse) and 12 weeks later were sacrificed, and the hαSyn expression was analyzed by immunofluorescence in the striatum using epifluorescence microscopy. Nuclei were stained with DAPI. Representative images of merged or single staining of hαSyn (red) or DAPI (blue) are shown. Scale bars are 100 µm. The insert on the upper-right corner of the merged images shows an area of interest in higher magnification. Please click here to view a larger version of this figure.
Figure 4: Loss of dopaminergic neurons of the SN in mice treated with different doses of AAV-hαSyn or control vector. Mice received AAV-hαSyn (1 x 1010 vg/mouse, 1 x 109 vg/mouse, or 1 x 108 vg/mouse) or AAV-GFP (1 x 1010 vg/mouse) and 12 weeks later were sacrificed, and TH was analyzed in the SNpc by immunohistochemistry. (A) Representative images. Scale bars, 100 µm. (B,C) The density of neurons was quantified as the number of TH+ neurons/mm2. Data represent mean ± SEM. n = 3–8 mice per group. (B) A comparison of ipsilateral with contralateral sides was performed using the two-tailed paired Student’s t-test. (C) A comparison of ipsilateral sides from mice receiving 1 x 1010 vg/mouse of AAV-hαSyn or AAV-GFP was carried out. (B,C) Whereas white bars indicate the quantification of TH+ neurons on the ipsilateral side of mice receiving AAV-hαSyn, green bars indicate the quantification of TH+ neurons on the ipsilateral side of mice receiving AAV-GFP. Grey bars indicate the quantification of TH+ neurons on the contralateral side of the corresponding group. Comparisons were performed by a two-tailed unpaired Student’s t-test. *p < 0.05; **p < 0.01. Please click here to view a larger version of this figure.
Figure 5: Analysis of the motor performance in mice treated with different doses of AAV-hαSyn. Mice received different doses (1 x 1010 vg/mouse, 1 x 109 vg/mouse, or 1 x 108 vg/mouse) of AAV-hαSyn or AAV-GFP, and 12 weeks later, the motor performance was evaluated by the beam test. (A) Image of a mouse walking on the beam. (B) The number of errors carried out by left limbs (contralateral) versus right limbs (ipsilateral) was quantified in the groups of mice receiving AAV-hαSyn. (C) The total number of errors was compared between different experimental groups receiving the same dose of AAV-hαSyn or AAV-GFP. Data represent mean ± SEM. n = 3–5 mice per group. Comparisons were performed by (B) a paired two-tailed Student’s t-test or by (C) an unpaired two-tailed Student’s t-test. *p < 0.05; **p < 0.01. Please click here to view a larger version of this figure.
Setting up the kinetics of the Parkinson's disease model induced by AAV-αSyn
After determining the proper AAV-hαSyn dose used to induce a significant level of neurodegeneration and motor impairment, experiments to define the onset of hαSyn overexpression were conducted. For this purpose, mice were treated with 1 x 1010 vg/mouse of AAV-hαSyn or sham surgery. The extent of hαSyn expression was analyzed in the SN once per week during weeks 2-5 after the stereotaxic surgery (see the experimental design in Supplementary Figure 2). The results show that, despite hαSyn expression being detected at low levels as early as 2 weeks after the surgery, hαSyn clusters appeared at week 5 after the stereotaxic surgery (Figure 6).
Figure 6: Analysis of the time course of human α-synuclein expression in the SN of mice treated with AAV-hαSyn. Mice received AAV-hαSyn (1 x 1010 vg/mouse) or just the sham stereotaxic surgery, and the expression of hαSyn in the SN was analyzed (A) 2 weeks, (B) 3 weeks, (C) 4 weeks, or (D) 5 weeks later by immunofluorescence using epifluorescence microscopy. Nuclei were stained with DAPI. Representative images of merged or single staining of hαSyn (red) or DAPI (blue) are shown. Scale bars, 100 µm. The insert on the upper-right corner of the merged images shows an area of interest in higher magnification. Please click here to view a larger version of this figure.
Afterward, experiments were conducted to determine the suitable time points to analyze neuroinflammation and T-cell infiltration in the central nervous system (CNS) after the stereotaxic delivery of AAV-hαSyn. To determine the peak of microglial activation after the treatment of mice with AAV-hαSyn, the extent of cells expressing high levels of Iba1 in the striatum was evaluated once per week during weeks 2-15 after the stereotaxic surgery. The results show a significant increase in microglial activation of the ipsilateral side compared with the contralateral side of mice 15 weeks after the AAV-hαSyn treatment (Figure 7). The number of Treg (CD4+ Foxp3+) cells infiltrated into the SNpc was also evaluated at different time points after the stereotaxic delivery of AAV-hαSyn by immunofluorescence followed confocal microscopy observation. The results show that the peak of Treg infiltration into the SNpc was at 11 weeks after surgery, whereas the extent of Treg infiltrating this area of the brain was lower at week 8 or week 13 after surgery (Figure 8). No CD4+ T cells were detected infiltrating the striatum (data not shown). Altogether, these results indicate that, using 1 x 1010 vg/mouse of AAV-hαSyn, the most suitable time point to analyze neuroinflammation is week 15 after the stereotaxic surgery, while a proper time point to analyze T-cell infiltration into the CNS seems to be week 11 after AAV-hαSyn treatment.
Figure 7: Analysis of the time course of microglial activation in mice inoculated with AAV-hαSyn. Mice received AAV-hαSyn (1 x 1010 vg/mouse), and microglial activation was evaluated by immunohistochemical analysis of Iba1 in the striatum at different time points after the surgery. (A) Representative overview images of immunohistochemical analysis of Iba1 from mice sacrificed 5 weeks, 10 weeks, or 15 weeks after inoculation with AAV-hαSyn are shown. Scale bars, 110 µm. (B) The density of activated microglia was quantified as the number of cells expressing high levels of Iba1 and ameboid shape per area. Data represent mean ± SEM. n = 3 mice per group. A two-tailed paired Student’s t-test was used to determine statistical differences between ipsilateral and contralateral Iba1 in each group. *p < 0.05. Please click here to view a larger version of this figure.
Figure 8: Analysis of the time course of CD4+ T-cell infiltration into the SN of mice inoculated with AAV-hαSyn. Foxp3gfp reporter mice received AAV-hαSyn (1 x 1010 vg/mouse). The presence of CD4+ T-cells expressing Foxp3 and the presence of TH+ neurons were analyzed at different time points (week 8, week 11, and week 13 after the surgery) in the SN by immunofluorescence. (A) Representative images for single immunostaining or the merge are shown. Scale bars, 100 µm. (B) The number of CD4+ Foxp3+ T cells per area in the SN was quantified. Data represent mean ± SEM from 3 mice per group. *p<0.05, ipsilateral versus contralateral CD4+ Foxp3+ T cells by two-tailed Student’s t-test. Please click here to view a larger version of this figure.
Supplementary Figure 1: Experimental design for evaluating the effect of different doses of AAV vectors on synuclein pathology, neurodegeneration, and motor impairment. Wild-type male C57BL/6 mice were anesthetized and received stereotaxic inoculation of different doses (1 x 1010 vg/mouse, 1 x 109 vg/mouse, or 1 x 108 vg/mouse) of AAV encoding human α-synuclein (AAV-hαSyn) or eGFP (AAV-GFP) under the control of the CBA promoter into the right substantia nigra (SN). After 12 weeks, the expression of GFP and hαSyn in the SN and striatum (Str) were evaluated by immunofluorescence (IF), tyrosine hydroxylase positive (TH+) cells were quantified by immunohistochemistry (IHC) in the SN, and motor performance was evaluated by the beam test. The number of mice in each experimental group is indicated in parenthesis. * indicates groups where one mouse died before the analyses. Each analysis indicates in parenthesis the number of the figure from the body of the paper where the corresponding results are shown. Please click here to download this File.
Supplementary Figure 2: Experimental design for determining the kinetics of T-cell infiltration, neuroinflammation, and hαSyn expression. Foxp3gfp reporter mice were anesthetized and received stereotaxic inoculation of AAV (1 x 1010 vg/mouse) encoding human α-synuclein (AAV-hαSyn) under the control of the CBA promoter into the right substantia nigra (SN) or sham surgery (PBS). Mice were sacrificed at different time points, and the expression of hαSyn in the SN and striatum was evaluated by immunofluorescence (IF), GFP (Foxp3), CD4, and tyrosine hydroxylase positive (TH+) cells were quantified by IF in the SN, and Iba1 expression was analyzed by immunohistochemistry (IHC) in the striatum (Str). The number of mice in each experimental group is indicated. The range of time points included in each analysis is indicated. Each analysis indicates in parenthesis the number of the figure from the body of the paper where the corresponding results are shown. Please click here to download this File.
The mouse model of neurodegeneration analyzed here might help study many critical aspects involved in the pathophysiology of Parkinson's disease, including the mechanisms involved in αSyn pathology and microglial activation, the involvement of the peripheral immune system in the regulation of neuroinflammation, and the mechanisms of neurodegeneration. Among the mechanisms involved in αSyn pathology are those subcellular mechanisms associated with mitochondrial, lysosomal, or proteasomal dysfunction in the presence of an excessive load of αSyn in the dopaminergic neurons of the SN2. It is important to consider that, in addition to the hαSyn expression induced by AAV-mediated transduction, the endogenous mouse αSyn also contributes to the load of total αSyn expression. Transgenic mice over-expressing mouse αSyn develop similar synuclein pathology, neuropathology, and motor impairment to those mouse models based on the overexpression of hαSyn32. Regarding microglial activation, the present mouse model might be used to study how different molecular and cellular players such as cytokines, neurotransmitters, astrocytes, neurons, the blood-brain barrier, and T cells might regulate the acquisition of pro-inflammatory or anti-inflammatory functional phenotypes8,10,11. This model also constitutes an important tool for studying the role of the peripheral immune system, including not only T cells but also macrophages, monocytes, and neutrophils, on the processes of neuroinflammation and neurodegeneration of nigral neurons11,33,34. Finally, this mouse model also represents a valuable system to study the cellular and molecular mechanisms of neurodegeneration in vivo, including those induced by internal cellular processes, such as oxidative stress, energy deficits, and damaged organelles 2, or those exerted by external players, such as neurotoxic factors produced by microglial cells, astrocytes, and cytotoxic T cells8,28,29,35.
A limitation of this mouse model is the study of how the pathological aggregation of αSyn in extra-cerebral locations might constitute the initial stages in the development of Parkinson’s disease36. In this regard, there is growing evidence indicating that, before the neurodegeneration of nigral neurons and motor impairment, αSyn pathology begins in the gut mucosa and the olfactory epithelium36 and, probably, the αSyn-specific T-cell response as well12. Afterward, αSyn aggregates would migrate through the vagus nerve to the brain stem, triggering the neuroinflammation and neurodegeneration of dopaminergic neurons12. Although the AAV-hαSyn model recapitulates most aspects of Parkinson’s disease, there is no evident involvement of the pathological aggregation of αSyn in extra-cerebral locations in this model. An alternative model involving hαSyn pathology appropriate for studying these aspects of Parkinson’s disease might be transgenic mice overexpressing hαSyn under the control of the Thy1 promoter, the Thy1-SNCA model37, in which disease development is dependent on the gut microbiota and involves an evident gastrointestinal impairment38.
Although it is helpful for the study of the diverse processes associated with the pathophysiology of Parkinson’s disease, the present mouse model involves critical steps that should be minutely checked, including the correct delivery of the viral vectors in the corresponding spatial coordinates, the selective expression of hαSyn in neurons (which depends on the AAV serotype and the vector construct), and the proper AAV dose and timing before analyzing the Parkinsonian phenotype. The analysis of the proper delivery of the viral vectors in the SN is necessary, as the usage of the correct spatial coordinates of the SN might not be enough when the needle is not entirely straight, which is sometimes imperceptible to the human eye. Moreover, the diffusion of the AAV vectors depends on the AAV serotype39. For these reasons, it is necessary to perform periodical quality controls checking the correct delivery and diffusion of the injected AAV-GFP vectors following the observation of GFP in brain slices containing the area of the SN.
Regarding the selective expression of hαSyn in neurons, in principle, the expression of hαSyn could be engineered to be controlled by a promoter selective for neurons or, even more precise, selective for dopaminergic neurons, such as the use of the TH promoter in AAV vectors to induce the selective expression of genes in dopaminergic neurons40. However, this strategy does not work when what is sought is overexpression of the gene of interest. For this reason, in the present model, it is essential to use a strong promoter (a promoter inducing high expression of the downstream gene) and AAV serotypes with neuronal tropism. In this study, the CBA promoter was used as a strong promoter to induce the overexpression of hαSyn, and the AAV5 serotype was used for the viral vector. This serotype has been used before to transduce mouse and rat neurons41,42. Here, the results demonstrated that, 12 weeks after the delivery of AAV5-GFP in the SN of mice, the green fluorescence was selectively present on the ipsilateral side of both the SN and striatum (Figure 1), indicating the efficient transduction of neurons of the nigrostriatal pathway.
Another critical aspect of this mouse model of Parkinson’s disease is the time point required to analyze a particular process after surgery. In this regard, this work shows a kinetic study of different processes involved in the pathology. Since key time points change with the dose of viral genomes given per mouse, the serotype of AAV used, or even with the batch of AAV used, a dose-response analysis of the amount of AAV-αSyn required to induce a significant loss of TH+ neurons and motor impairment was first carried out. Previous studies have shown significant motor impairment and a loss of TH+ neurons of the nigrostriatal pathway after 12 weeks of AAV-αSyn injections in mice at doses ranging from 6 x 108–3 x 1010 viral genomes per mouse16,17,30,31. Accordingly, the dose of AAV-hαSyn used to induce the hαSyn expression in the nigrostriatal pathway, the loss of TH+ neurons, and motor impairment in mice ranged from 1 x 108–1 x 1010 viral genomes per mouse. Moreover, to control that the loss of TH+ neurons and motor impairment were induced by the overexpression of hαSyn in the SN and not by AAV infection of neurons of the SN, control groups were included in which AAV coding for a reporter gene (AAV-eGFP) was delivered unilaterally in the SN of mice and neurodegeneration and motor impairment were determined. The results showed that, 12 weeks after the stereotaxic surgery, 1 x 1010 viral genomes per mouse was a proper dose of AAV5-hαSyn, as mice receiving this viral load displayed significant hαSyn in the nigrostriatal pathway (Figure 2 and Figure 3), loss of TH+ neurons (Figure 4), and motor impairment (Figure 5). In contrast, lower doses of AAV5-hαSyn (1 x 108 viral genomes per mouse and 1 x 109 viral genomes per mouse) were not strong enough to reach significant changes in all these parameters together (Figures 2–4). Of note, the administration of AAV-GFP at 1 x 1010 viral genomes per mouse induced a low (~20%), but significant degree of loss of TH+ neurons of nigral dopaminergic neurons (Figure 4A,B). This result agrees with previous observations using this model41 and is probably the consequence of a low level of neuroinflammation induced by the administration of AAV vectors in the SN. Nevertheless, the extent of loss of TH+ neurons was significantly higher in mice receiving AAV5-hαSyn compared with those receiving the same dose of AAV-GFP (Figure 4C). Of note, the kinetics of hαSyn expression not only depend on the efficiency of transduction but also on the extent of AAV diffusion39. Since AAV diffusion depends on the AAV serotype, the precise key time points in this animal model may vary when using another AAV serotype different from AAV5.
Afterward, a kinetic analysis was conducted using 1 x 1010 viral genomes per mouse to determine key time points in this mouse model. Since current evidence has shown some early symptoms that appear before motor impairment, which would allow the early diagnosis of Parkinson’s disease43,44, these experiments sought to find the time point at which hαSyn expression was already evident but in the absence of motor impairment. The results show that the onset of hαSyn expression in the SN was at 5 weeks after the stereotaxic delivery of AAV-hαSyn (Figure 6). This time point constitutes an interesting temporal point to start administering therapies tailored to stop the neuroinflammatory and neurodegenerative processes. Other key time points determined here were the peak times for two critical events associated with the neuroinflammation process: the time at which microglia reach the maximal degree of activation and the time of maximal T-cell infiltration into the SN. The results showed a curve with a trend reaching two waves of maximal microglial activation, the first one at 10 weeks after the surgery and the second one at 15 weeks after the surgery (Figure 7). The kinetic analysis of T-cell infiltration showed the peak time of Treg infiltration into the SN at 11 weeks after the stereotaxic surgery (Figure 8). Surprisingly, no effector T cells (CD4+ Foxp3–) were detected infiltrating the SN during the time frame analyzed (weeks 8–13 after surgery). Altogether, these results suggest a proper frame of time to start administering therapies geared toward stopping the process of neuroinflammation and attenuating T-cell infiltration into the SN using this preclinical model, which ranges between week 5 after surgery (the onset of hαSyn overexpression) and week 10 after surgery (the first wave of neuroinflammation and T-cell infiltration) (Figure 9).
Figure 9: Summary of the key time points found for this animal model. Please click here to view a larger version of this figure.
The authors have nothing to disclose.
We thank Dr. Sebastián Valenzuela and Dr. Micaela Ricca for their valuable veterinary assistance in our animal facility. This work was supported by "Financiamiento Basal para Centros Científicos y Tecnológicos de Excelencia de ANID" Centro Ciencia & Vida, FB210008 (to Fundación Ciencia & Vida), and the Geroscience Center for Brain Health and Metabolism, FONDAP-15150012. This work was also funded by grants FONDECYT-1210013 (to R.P.) and FONDECYT-1150766 (to F.C.) from "Agencia Nacional de Investigación y Desarrollo de Chile (ANID)" and MJFF-10332.01 (to R.P.) and MJFF-17303 (to F.C.) from the Michael J Fox Foundation for Parkinson's Research.
ANIMALS AND ANIMAL FOOD | |||
Foxp3-GFP C57BL/6 mice | The Jackson Laboratory (Bar Harbor, ME) | Stock No: 023800 | |
Laboratory Rodent Diet | LabDiet | Rodent Diet 5001 | Standard Rodent diet |
Wild-type C57BL/6 mice | The Jackson Laboratory (Bar Harbor, ME) | Stock No: 000664 | |
VIRAL VECTORS | |||
AAV5-CBA-αSyn | University of Iowa Viral Vector Core Facility | N/A | Stock concentration at 10E13 vg/mL |
AAV5-CBA-eGFP | University of Iowa Viral Vector Core Facility | N/A | Stock concentration at 9.5 x 10E12 vg/mL |
ANESTHETICS AND ANALGESICS | |||
Isoflurane | Baxter | 218082 | 1% for stereotaxic surgery |
Ketamine | Drag Pharma | CHE30 | 70 mg/Kg for stereotaxic surgery |
Sevoflurane | Baxter | VE2L9117 | For before transcardial perfusion |
Tramadol | Drag Pharma | DPH134 | 30 mg/Kg every 24 h |
Xylazine | Centrovet | EHL40 | 9 mg/kg for stereotaxic surgery |
EQUIPMENT | |||
Beam test | Home made | N/A | horizontal beam 25 cm length and 3 cm width. The beam surface was covered by a metallic grid (1 cm2). |
Cryostate | Leica | CM1520 | |
Digital camera | Nikon | S2800 Coolpix | For recording the beam test performance |
Microscope | Olympus | BX51 | Used for IHC analysis (section 4.4) |
Microscope | Olympus | IX71 | Used for IF analysis (section 5.3) |
Microscope | Leica | DMI8 | Used for IF analysis (section 5.7) |
New Standard Stereotaxic, mouse | Stoelting, Wood Dale, IL, USA | 51500 | stereotaxic frame for surgery |
Peristaltic Pump | Masterflex | C-flex L/S16 | |
Power supply unit | Olympus | U-RFL-T | Used for IF analysis (section 5.3) |
Surgical suture | Sylkam®, B Braun | C0760171 | |
Syringe 100 U | BD | 324918 | For anesthesia before transcardial perfusion, 29G needle |
Syringe RN 5uL SYR W/O NEEDLE | Hamilton | HA-7641-01 | For viral vector innoculation |
BUFFERS AND REAGENTS | |||
Aviden, Peroxidase Conjugate | Merck, Darmstadt, Germany | 189728 | |
Bovine Serum Albumin | Merck, Darmstadt, Germany | 9048-46-8 | |
Cryotrotection buffer | Home made | N/A | 20% glycerine and 2% DMSO in PBS |
DAPI | Abcam | ab228549 | |
Diaminobenzidine | Merck, Darmstadt, Germany | D8001 | |
Fluoromount -G T | Electron Microscopy Science | 17984-25 | |
Gelatin | Merck, Darmstadt, Germany | 104078 | |
Normal goat serum | Jackson ImmunoResearch Laboratory | 5000121 | |
Paraformaldehyde | Merck, Darmstadt, Germany | 104005 | |
PBS | Home made | N/A | 0.125 M, pH 7.4 |
Peroxidase inactivating buffer | Home made | N/A | 0.03% H2O2 in methanol |
Triton X-100 | Sigma-Aldrich | 9036-19-5 | |
Trizma Hydrochloride | Merck, Darmstadt, Germany | 1185-53-1 | |
Tween 20 | Sigma-Aldrich | 822184 | |
ANTIBODIES | |||
Biotin-SP (long spacer) AffiniPure Goat Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch Laboratory | 111065003 | |
Goat anti-Rabbit IgG (H+L) Alexa Fluor 546 | ThermoFisher Scientific | A11010 | |
Goat anti-Rabbit IgG (H+L) Alexa Fluor 647 | ThermoFisher Scientific | A21244 | |
Goat anti-Rat IgG (H+L) Alexa Fluor 546 | ThermoFisher Scientific | A11081 | |
Rabbit monoclonal anti-alpha-Synuclein | Abcam | ab138501 | |
Rabbit monoclonal anti-Iba-1 | Abcam | EPR16588 | |
Rabbit polyclonal anti-Tyrosine Hydroxylase | Millipore | AB152 | |
Rat monoclonal anti-CD4 | Biolegend | 100402 | |
SOFTWARES | |||
GraphPad | Prism | 6.0 | Fos stats analysis |
ImageJ | National Institute of Health | N/A | For image analysis |
LAS X | Leica | N/A | For image capture with Leica microscope |
ProgRes Capture Pro | Jenoptik | N/A | For image capture with Olympus microscope |
VLC media player | VideoLAN Organization | N/A | For analysis of behavioural tests |