Parkinson's Disease

Q: What are key differences between PFF and AAV injected Parkinson's mice models?

While both types of models share several common features, such as robust neuroinflammation and neurodegeneration, there are specific differences. Based on the particular target(s) and mechanism(s)-of-action of a therapeutic agent, one model may possess advantages over the other. PFF-injected animals demonstrate spreading of misfolded α-synuclein fibrils, which is thought to be a key pathologic process in neurodegenerative diseases, including Parkinson’s disease. The specific phenotype depends on the injection site (neuroanatomical pathways targeted) and disease duration. These animals can develop widespread pathology throughout the brain (including dopaminergic and non-dopaminergic neurons) with a well-defined spatiotemporal pattern in cortical and subcortical structures, thereby replicating multiple motor and non-motor aspects of human PD. AAV-injected animals develop a strong phenotype associated with unilateral dopaminergic deficits. Behaviorally, these mice are similar to 6-hydroxydopamine (6-OHDA) lesioned animals, with the advantage that the pathology is driven by α-synuclein, rather than by a chemical toxin, thereby providing greater clinical relevance. These mice show selective degeneration of dopaminergic neurons in the substantia nigra pars compacta and concomitant loss of dopaminergic terminals in the striatum. Highly significant motor deficits can be readily measured in these mice (e.g. rotarod test) within a relatively short timeframe (2-3 months). Unilateral stereotaxic injection of AAV A53T α-synuclein vectors induces progressive loss of dopaminergic neurons in the substantia nigra pars compacta and associated motor and reflex deficits (e.g. swinging to the contralateral side). Note that pigmented neurons are shown only for illustrative purposes (mice do not have pigmented neurons in the substantia nigra

Q: What are the advantages of injecting PFFs into transgenic mice?

Transgenic mice overexpressing human α-synuclein have higher levels of α-synuclein expression in neurons compared to non-transgenic animals. As such, there is more “endogenous” α-synuclein “substrate” available for induced misfolding as part of the pathology spreading process. As a result, a greater level of α-synuclein aggregates can be observed in cell bodies and processes compared to that observed in wild-type mice. This greater burden of α-synuclein pathology leads to significant neuroinflammation (with activated microglia and reactive astrocytes) and neurodegeneration. As a consequence, distinct phenotypes with altered behavior, brain atrophy, changes in fluid biomarkers, etc., which can serve as robust readouts in preclinical efficacy studies, can be observed in these PFF-injected transgenic animals.

Q: How can neurodegeneration be measured in α-synuclein animal models?

In PFF-induced models, which show extensive neurodegeneration in multiple brain regions, in-life measures, such as MRI-based brain atrophy and blood & CSF neurofilament light chain, can be used. An advantage of these measures is that they can serve as clinically translation biomarkers. Read more about these measures in Brain Atrophy Analysis in Mouse Models of Neurodegeneration and Neurofilament Light Chain in Parkinson's Disease Models. In AAV-induced models, which demonstrate neurodegeneration of dopaminergic neurons localized to the substantia nigra and striatum, quantification of dopaminergic neuron nuclei, cell bodies, cell processes (axons, dendrites), and synaptic terminals can be readily achieved via immunohistochemistry or multiplex immunofluorescence. Examples can be seen in AAV A53T α-Synuclein Mouse Model of Parkinson's Disease and AAV α-Synuclein Models for Parkinson's Disease Drug Development.

Q: Can non-motor symptoms be observed in α-synuclein mouse models?

Non-motor symptoms are key features of human Parkinson’s disease. Depending on the injection site of the PFFs in transgenic α-synuclein-overexpressing mice, non-motor symptoms (e.g. sleep alterations, reduced olfaction, behavioral & cognitive deficits) can be found. For example, by targeting the limbic system, highly significant, progressive changes in sleep-wake cycles can be observed and quantitatively measured.

α-Synuclein Preformed Fibril (PFF) Models

The pathologic spread of misfolded alpha-synuclein that is characteristic of human Parkinson's disease can be modelled in animal brains by injection of α-synuclein preformed fibrils (PFFs). This "PFF seeding & spreading model" can be induced in transgenic mice overexpressing human α-synuclein or in wild-type mice or rats.

This highly reproducible animal model of synucleinopathy replicates several key feature of human PD, including α-synuclein aggregates in the cell body and neurites, neurodegeneration (measurable via neurofilament light chain in the blood & CSF, as well as by in vivo MRI-based brain atrophy measures), microgliosis, astrogliosis, and dopaminergic denervation. Motor deficits and alterations of sleep architecture can also be quantitatively measured in this model.

MORE INFORMATION ON α-SYNUCLEIN PFF MODELS

A pair of brain tissue sections processed with immunohistochemistry (IHC) to highlight phosphorylated alpha-synuclein, which is relevant in the context of Parkinson's Disease research

AAV A53T α-Synuclein Mouse Models

Generation of alpha-synuclein pathology in the adult rodent brain can be generated via injection of adeno-associated virus (AAV) vectors. In this mouse model of Parkinson's disease, wild-type (C57BL/6) mice undergo stereotaxic injection of AAV vectors overexpressing A53T mutant human alpha-synuclein into the vicinity of the substantia nigra pars compacta.

This robust synuclein model pathologically shows synuclein aggregates in the neuron soma and neurites, neuroinflammation (including activated microglia and reactive astrocytes), neurodegeneration, and dopaminergic denervation. Significant motor deficits are observed in these Parkinson's mice models resulting from the unilateral dopaminergic neuron loss, including alterations in the rotarod test, cylinder test, tail suspension swing test, and hindlimb clasping test.

MORE INFORMATION ON AAV α-SYNUCLEIN MODELS

Dopaminergic denervation of ipsilateral striatum shown by tyrosine hydroxylase immunostaining

Translatability of our Parkinson's Disease Models to Human Disease

Alpha-Synuclein Aggregates

Aggregates of misfolded α-synuclein is a key pathologic feature of human Parkinson's disease. Lewy bodies and Lewy neurites are observed in dopaminergic neurons in the substantia nigra pars compact and other brain regions. Misfolded α-synuclein pathology also follows a spatiotemporal pattern (Braak, 2003). We observe high levels of phosphorylated α-synuclein in neuronal soma and processes in both our AAV-induced and preformed fibril (PFF)-induced models. We also have robust seeding and spreading in the PFF models.

Activated Microglia & Reactive Astrocytes

Neuroinflammation is a key pathologic feature of Parkinson's disease. Activated microglia and reactive astrocytes a play a key role in pathogenesis (Kam, 2020; Chen, 2023). We have found distinct spatiotemporal patterns of neuroinflammation in our AAV- and PFF-induced mouse models. We have also shown alterations of microglial and astrocytic morphology in these models using algorithms based on computer vision and machine learning that we have developed.

AAV - EBST Test Results (Box and Whiskers)

Dopaminergic Neuron Loss & Motor Deficits

Extrapyramidal motor symptoms are a central clinical feature of Parkinson's disease. Motor dysfunction results from loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and denervation of the striatum (e.g. caudate and putamen). By targeting the SNc with AAVs overexpressing α-synuclein or with α-synuclein PFFs, we have shown neurodegeneration of dopaminergic neurons and loss of dopaminergic terminals in our models. These mice show alterations of motor function by various tests, including the Tail Suspension Swing Test, Cylinder Test, Hindlimb Clasping Test, and Rotarod Test.

Sleep Alterations

Sleep disturbances are prevalent non-motor symptoms of Parkinson's disease (Stefani and Högl, 2020) which affects up to ~85% of patients (Asadpoordezaki, 2025). Using a non-invasive system for assessment of sleep in mice, we have reproducibly demonstrated alterations of sleep-wake architecture (e.g. percentage of sleep, sleep bout lengths) when α-synuclein PFFs are injected into anterior olfactory nucleus (AON) of transgenic mice overexpressing human A53T mutant α-synuclein.

Regional Brain Atrophy

Brain imaging biomarkers are widely used in clinical trials of neurodegenerative diseases, including Parkinson's disease. MRI-derived regional volume and cortical thickness measures are highly sensitive to brain atrophy in PD. It has been shown that the progression MRI-based brain atrophy in Parkinson's disease is consistent with the prion-like propagation hypothesis of α-synuclein (Tremblay, 2021; Abdelgawad, 2023). Using high-resolution, whole brain MRI acquisition and fully-automated image processing & analysis, we have shown reproducible regional brain atrophy in both PFF- and AAV-based PD models, thereby serving as a robust in-life measure of neurodegeneration.

Elevated Neurofilament Light in CSF & Plasma

Neurofilament light chain is increased in the CSF and plasma of PD patients (Bäckström, 2020; Urso, 2023; Pedersen, 2024). Neurofilament light measurements are routinely used in PD clinical trials. Elevated neurofilament light chain levels have been observed in several animal models of PD. We observe highly significant increases in plasma & CSF levels of neurofilament light in our mouse models with human α-synuclein PFFs injected into the anterior olfactory nucleus (AON) or medial forebrain bundle (MFB) of M83+/- transgenic mice.

Parkinson's Disease Mouse Models Features

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The Interactive Presentation below allows you to explore our characterization of our AAV-Synuclein mouse model, including in vivo data and high-resolution images of entire Multiplex Immunofluorescence tissue sections.

You can simply navigate through this "Image Story" using the left panel.

You can pan around the high-resolution microscopy images using the left mouse button. You can zoom in and out using the mouse/trackpad (up/down) or the + and - buttons in the upper left corner. You can toggle (on/off), change color, and adjust image settings for the channels and segmentations in the Control Panel in the upper right corner.

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Neurodegeneration & Neuroinflammation in the AAV-Synuclein Mouse Model

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This Interactive Presentation illustrates some of the interesting motor function, brain imaging, and pathologic features of Biospective's AAV A53T α-synuclein mouse model of Parkinson’s disease (PD).

This model was generated by injecting 12 week-old C57BL/6 mice with AAV-human-A53T-synuclein or AAV-null (control) vectors unilaterally into the left substantia nigra pars compacta (SNc). 2 µL of vector was infused at a rate of 0.4 µL/min using a digital stereotaxic device with an automated microinjector.

Coronal Image of Mouse Brain with AAV Injection Site in the SNc

Coronal Atlas View of SNc Injection Site

Multiplex immunofluorescence (mIF) images were generated by immunostaining for phospho-Syn129, GFAP, Iba-1, Tyrosine Hydroxylase, Dopaminergic Nuclei, and counterstained with the DAPI nuclear stain. Tissue sections were digitized using a high-throughput slide scanner and were processed using Biospective's PERMITS^TM software platform.

To navigate though this Image Story, you can use the arrows and/or the Table of Contents icon in the upper right corner of this panel.

You can also interact with the microscopy image in the viewer on the right at any time to further explore this high-resolution data.

Neurodegeneration in the Substantia Nigra

As can be seen in this microscopy image, there is substantial loss of TH-positive dopaminergic neurons in the ipsilateral SNc compared to the contralateral hemisphere. For reference, an illustration with atlas labels for this brain level is provided below.

Coronal Mouse Brain Section (Bregma -3.2) with Neuroanatomy Labels

Using our PERMITS^TM quantitative analysis software, we have quantified the TH staining in the SNc. The plots below show a highly significant reduction in the ipsilateral hemisphere.

Tyrosine Hydroxylase and Cell Density in the Substantia Nigra

TH stain density and cell density for AAV-Syn compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

We have found significant brain atrophy in the SNc by generating regional volume data from in vivo anatomical MRI scans, which corresponds well with the loss of TH-positive neurons. MR images were acquired from mice injected with different doses of AAV-Synuclein at 4 weeks post-inoculation using a 7T animal MRI scanner.

Anatomical MRI with segmented SNc, as well as a plot of relative difference between ipsilateral and contralateral SNc. Injected AAV-Syn doses (GC) were 1×10⁹ (yellow), 5×10⁹ (blue), and 1×10¹⁰ (aqua). *p<0.05, **p<0.01.

Dopaminergic Neurons in the Contralateral SNc

This microscopy image shows the contralateral (right hemisphere) SNc which shows unaffected TH-positive cell bodies and processes in red. The DAPI-counterstained nuclei are shown in blue.

Loss of Dopaminergic Neurons in the Ipsilateral SNc

This microscopy image shows the ipsilateral (left hemisphere) SNc, which demonstrates a substantial reduction of TH-positive cell bodies and processes (in red) compared to the contralateral hemisphere. The DAPI-counterstained nuclei are shown in blue.

Neurodegeneration in the Caudate-Putamen & Dopaminergic Motor Deficits

This microscopy image show severe dopaminergic denervation of the ipsilateral (left hemisphere) caudate-putamen (loss of TH-positive terminals). For reference, an illustration with atlas labels for this approximate brain level is provided below.

Coronal Image of Mouse Brain at the Level of the Striatum

Coronal Mouse Brain Section (Bregma +0.86) with Neuroanatomy Labels

Using our PERMITS^TM quantitative analysis software, we have quantified the TH staining in the Caudate-Putamen. The plot below shows a highly significant reduction in the ipsilateral hemisphere.

Tyrosine Hydroxylase Staining in the Caudate-Putamen

TH stain density for AAV-Syn compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

This loss of dopaminergic innervation corresponds well with unilateral motor deficits in these mice, including a highly significant increase in use of the ipsilateral paw during the Cylinder Test, decreased latency to fall in the Rotarod Test, and increased swings to the contralateral side in the Tail Suspension Swing Test (TSST).

Illustration of Motor Tests and Plots of AAV-Syn vs. AAV-null

Cylinder Test, Rotarod Test, and Tail Swing Suspension Test (TSST) data for AAV-Syn compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

Loss of Dopaminergic Terminals in the Ipsilateral Caudate-Putamen

This high magnification view shows the severe extent of loss of dopaminergic (TH-positive) terminals in the ipsilateral striatum. There are some remaining (albeit dystrophic) axons present.

Similar to our findings in the SNc, we have observed brain atrophy in the caudate-putamen by quantitative analysis of high-resolution anatomical MRI scans, which establishes an in vivo-ex vivo relationship between neuroimaging and IF measures.

MRI Atlas and Volume Data at the Level of the Striatum

Anatomical MRI with segmented caudate-putamen, as well as a plot of the relative difference between ipsilateral and contralateral caudate-putamen. Injected AAV-Syn doses (GC) were 1×10⁹ (yellow), 5×10⁹ (blue), and 1×10¹⁰ (aqua). *p<0.05, **p<0.01.

Microgliosis in Response to Human A53T a-Synuclein Expression

In this low magnification image, one can readily appreciate the higher density of Iba-1 staining microglia in the ipsilateral hemisphere (indicated by the box) relative to the contralateral hemisphere in an AAV-Syn injected mouse brain.

The plots below show the Iba-1 stain density in various brain regions, with highly significant increased staining in the AAV-Syn mice.

Iba-1 stain density for AAV-Syn compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

We have performed a morphological analysis of microglia using a novel computer vision & machine learning approach developed by our team. This fully-automated algorithm classifies non-activated (ramified) and activated (non-ramified) microglia.

Examples of Non-activated and Activated Microglial Morphology

The plots below show the microglial activation in various brain regions, with highly significant increased microglial activation in the AAV-Syn mice.

Plots of PERMITS Data Showing Activated Microglia

Microglial activation for AAV-Syn compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

Iba-1 Staining in Proximity to Phosphorylated α-Synuclein

This high magnification view shows the increased density of Iba-1-stained microglia in areas with phosphorylated α-synuclein aggregates.

Astrogliosis in Response to Human A53T α-Synuclein Expression

This low magnification microscopy image show a higher density of GFAP-positive astrocytes in the ipsilateral hemisphere (indicated by the box) of an AAV-Syn injected mouse brain. The plots below show the GFAP stain density in various brain regions.

Plots of PERMITS Data Showing GFAP Stain Density

GFAP stain density for AAV-Syn compared to AAV-null (control) injections; mean ± SEM, t-test, **** p<0.0001.

Astrogliosis and Microgliosis

This high magnification microscopy image shows a high level of Iba-1-positive microglia and GFAP-positive astrocytes in the ipsilateral hemisphere. Note the “activated” morphology of these neuroinflammatory cells.

Summary

The AAV A53T α-syn mouse model recapitulates many of the hallmark features of Parkinson’s disease. This model demonstrates progressive development of asymmetric motor dysfunction (due to unilateral injection), and associated loss of TH-positive SNc neurons and striatal TH expression.

AAV A53T α-synuclein locally increases brain atrophy, microglial density and activation levels, and astrocyte density and hypertrophy. Studies are planned to further interrogate the spatial relationships between microglial activation, astrocyte hypertrophy, and α-synuclein aggregation.

The AAV A53T α-synuclein mouse model is well-suited for drug development given the quantitative in-life and ex vivo readouts. It also has advantages over other models as a screening tool for novel disease-modifying therapeutics targeting α-synuclein related pathology, including the relatively short timeframe required to perform preclinical studies in this model.

Please feel free to further explore the microscopy image in the viewer.

We would be happy to discuss this model and our characterization if you would like to Contact Us.

Table of Contents

Learn more about our characterization of our Parkinson's Disease mouse models, our validated measures, and our Preclinical Neuroscience CRO services.

FAQs

What are key differences between PFF and AAV injected Parkinson's mice models?

PFF-injected animals demonstrate spreading of misfolded α-synuclein fibrils, which is thought to be a key pathologic process in neurodegenerative diseases, including Parkinson’s disease. The specific phenotype depends on the injection site (neuroanatomical pathways targeted) and disease duration. These animals can develop widespread pathology throughout the brain (including dopaminergic and non-dopaminergic neurons) with a well-defined spatiotemporal pattern in cortical and subcortical structures, thereby replicating multiple motor and non-motor aspects of human PD.

AAV-injected animals develop a strong phenotype associated with unilateral dopaminergic deficits. Behaviorally, these mice are similar to 6-hydroxydopamine (6-OHDA) lesioned animals, with the advantage that the pathology is driven by α-synuclein, rather than by a chemical toxin, thereby providing greater clinical relevance. These mice show selective degeneration of dopaminergic neurons in the substantia nigra pars compacta and concomitant loss of dopaminergic terminals in the striatum. Highly significant motor deficits can be readily measured in these mice ( rotarod test) within a relatively short timeframe (2-3 months).

What are the advantages of injecting PFFs into transgenic mice?

Transgenic mice overexpressing human α-synuclein have higher levels of α-synuclein expression in neurons compared to non-transgenic animals. As such, there is more “endogenous” α-synuclein “substrate” available for induced misfolding as part of the pathology spreading process. As a result, a greater level of α-synuclein aggregates can be observed in cell bodies and processes compared to that observed in wild-type mice. This greater burden of α-synuclein pathology leads to significant neuroinflammation (with activated microglia and reactive astrocytes) and neurodegeneration. As a consequence, distinct phenotypes with altered behavior, brain atrophy, changes in fluid biomarkers, etc., which can serve as robust readouts in preclinical efficacy studies, can be observed in these PFF-injected transgenic animals.

How can neurodegeneration be measured in α-synuclein animal models?

In PFF-induced models, which show extensive neurodegeneration in multiple brain regions, in-life measures, such as and , can be used. An advantage of these measures is that they can serve as clinically translation biomarkers. Read more about these measures in and .

In AAV-induced models, which demonstrate neurodegeneration of dopaminergic neurons localized to the substantia nigra and striatum, quantification of dopaminergic neuron nuclei, cell bodies, cell processes (axons, dendrites), and synaptic terminals can be readily achieved via or . Examples can be seen in and .

Can non-motor symptoms be observed in α-synuclein mouse models?

Non-motor symptoms are key features of human Parkinson’s disease. Depending on the injection site of the PFFs in transgenic α-synuclein-overexpressing mice, non-motor symptoms ( sleep alterations, reduced olfaction, behavioral & cognitive deficits) can be found. For example, by targeting the limbic system, highly significant, progressive changes in sleep-wake cycles can be observed and quantitatively measured.

References

Keywords

proteins are composed of linear chains of amino acids that must fold into a functional three-dimensional structure. When these chains fail to fold properly, the resulting proteins can adopt abnormal shapes. These proteins are typically insoluble, and disrupt normal cellular processes. The accumulation of misfolded proteins is closely linked with several neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic lateral sclerosis (ALS).

a neurodegenerative disease that is characterized by: (a) movement-related (motor) symptoms, including resting tremor, bradykinesia, rigidity, and postural instability, related to the loss of dopaminergic neurons in the substantia nigra; and (b) non-motor symptoms including anxiety, depression, apathy, hallucinations, constipation, orthostatic hypotension, sleep disorders, hyposmia/anosmia, and cognitive impairment.