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Other Studies on Using ASOs in Treating Parkinson's

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Other Studies on Using ASOs in Treating Parkinson's

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Progressive neuronal death in brainstem nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Reduction of α-synuclein levels is therefore a potential therapy for PD. However, because α-synuclein is essential for neuronal development and function, α-synuclein elimination would dramatically impact brain function. We previously developed conjugated small interfering RNA (siRNA) sequences that selectively target serotonin (5-HT) or norepinephrine (NE) neurons after intranasal administration. Here, we used this strategy to conjugate inhibitory oligonucleotides, siRNA and antisense oligonucleotide (ASO), with the triple monoamine reuptake inhibitor indatraline (IND), to selectively reduce α-synuclein expression in the brainstem monoamine nuclei of mice after intranasal delivery. Following internalization of the conjugated oligonucleotides in monoamine neurons, reduced levels of endogenous α-synuclein mRNA and protein were found in substantia nigra pars compacta (SNc), ventral tegmental area (VTA), dorsal raphe nucleus (DR), and locus coeruleus (LC). α-Synuclein knockdown by ∼20%-40% did not cause monoaminergic neurodegeneration and enhanced forebrain dopamine (DA) and 5-HT release. Conversely, a modest human α-synuclein overexpression in DA neurons markedly reduced striatal DA release. These results indicate that α-synuclein negatively regulates monoamine neurotransmission and set the stage for the testing of non-viral inhibitory oligonucleotides as disease-modifying agents in α-synuclein models of PD.

Keywords

intranasal administration

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View Abstract

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Selective α-Synuclein Knockdown in Monoamine Neurons by Intranasal Oligonucleotide Delivery: Potential Therapy for Parkinson's Disease

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Progressive neuronal death in brainstem nuclei and widespread accumulation of α-synuclein are neuropathological hallmarks of Parkinson's disease (PD). Reduction of α-synuclein levels is therefore a potential therapy for PD. However, because α-synuclein is essential for neuronal development and function, α-synuclein elimination would dramatically impact brain function.

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Results: Incorporation of new DA neurons in nigrostriatal pathway

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Results: Incorporation of new DA neurons in nigrostriatal pathway

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Green fluorescent retrobeads were injected in CPu region 1-3 months after AAV-shPTB delivery

important in enabling axonal uptake and retrograde labelling for cell bodies.

1 day after,  green retrobeads were observed  in both TH+RFP- cells and TH+RFP+ cells in substantia nigra

1 month after, retrobeads were only observed in DA neurons

3 months after, retrobeads were observed in both TH+RFP- cells and newly converted TH+RFP+ cells

Ultimately, incorporation of new DA neurons in nigrostriatal pathway was found to be time-dependent

* arrows= TH+ DA neurons
* arrowheads= RFP+ cells
Image from Qian et al., (2020) Figure 3g and Figure 9g,h

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Results: Neuron conversion region specificity

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Results: Neuron conversion region specificity

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AAV-shPTB injected in midbrain, cortex, and striatum

Most RFP+TH+DA neurons found in midbrain

In cultured models, cortical astrocytes treated with lentiviral shPTB

Observed that in

Cortical- 2% became TH+

Midbrain- about 10% became TH+

RT-qPCR analysis conducted on isolated cortical and midbrain astrocytes

found that isolated midbrain astrocytes had higher levels of transcription factors compared to cortical astrocytes

Ultimately, observed that there is region specificity in terms of neuron conversion and that the midbrain region had a higher neuron conversion

Image from Qian et al.,Figure 3a,b and Figure 8e, f

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Results: Maturation of Newly converted Dopaminergic (DA) neurons

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Results: Maturation of Newly converted Dopaminergic (DA) neurons

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3- 12 weeks after AAV-shPTB injection, new DA neurons in cells were observed

Stained with DA markers : DOPA decarboxylase (DDC) and tyrosine hydroxylase (TH)

Patch clamp recordings were conducted on converted neurons

Converted neurons found to be continuously increasing

After 12 weeks, 30-35% of RFP+ cells were neurons

Observed hyperpolarization-active and cyclic nucleotide gated (HCN) channels activity and wider action potentials

After 6 weeks: no HCN activity

After 12 weeks: HCN activity visible

HCN channel activity and wider action potentials are characteristics of mature neurons entailing that newly converted DA neurons were progressively maturing
Image from  Qian et al.,Figure 2h

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AAV-shPTB and AAV-empty injected into substantia nigra of midbrain

AAV-shPTB

3 weeks after, 20% RFP+ cells expressed NeuN+

5 weeks after,  RFP+NeuN+ cells were 3 times greater than of 3 weeks after

10 weeks after, about 80% were RFP+ NeuN+ GFAP-

AAV-empty

10 weeks after, most RFP+ cells were astrocytes

Therefore, injection of AAV-shPTB into substantia nigra enabled SUCCESSFUL conversion from astrocytes to neurons
Image from Qian et al., (2020) Figure 2 e,f

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Results: Functionality of Converted Neurons

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Results: Functionality of Converted Neurons

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After 4 weeks, 50- 80% of shPTB transduced cells demonstrated neuronal phenotypes

cells stained positive for TUJ1+,MAP+, neuronal markers

Through patch clamp recording,

Na+/K+ channel current detected

Repetitive Action Potential Firing (AP) detected

Therefore, depletion of PTB enabled SUCCESSFUL conversion of astrocytes to functional neurons
Image from Qian et al., (2020) Figure 2a,b

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Results: PTB-ASO has the same effectiveness as AAV-shPT

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Results: PTB-ASO has the same effectiveness as AAV-shPT

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in vitro (mouse astrocytes) and in vivo (midbrain of mice) testing

In vitro

PTB-ASO: neuronal markers present after 5 weeks (TUJ1, MAP2, NSE, NeuN) - astrocytes differentiated to neurons

some were TH+ (DA neurons)

Patch clamp recordings showed neurons were functional

In vivo

PTB-ASO: tdTomato cells turned to NeuN+ (8 weeks) and to TH+ (12 weeks) -astrocytes differentiated to neurons and particularly DA neurons

Patch clamp recordings showed neurons were functional

injected 6-OHDA and conducted behavioural tests: showed motor improvement in same manner as AAV-shPTB on same three behavioural tests

ASO is a suitable alternative to the AAV vector - good for administering in clinical setting

Image from Qian, et al. (2020) - Figure 7 b-d, f-g

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Results: AAV sh-PTB causes reversal in behaviour -Chemogenetic analysis

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Results: AAV sh-PTB causes reversal in behaviour -Chemogenetic analysis

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6-OHDA mice

limb-use bias test

AAV-hM4Di-shPTB:

motor performance restored to WT levels after two months

following CNO injection: motor performance improvement disappeared (Parkinson's disease phenotype restored) until CNO metabolized fully (72 hours)

following saline injection: no change - still beneficial

AAV-hM4Di-empty:

no benefit initially or following CNO/ saline injection

The DA neurons induced by the AAV-shPTB vector was ultimately the main source that was allowing behavioural reversal back to WT

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Results: Fixing Parkinson's Disease Motor Behaviours

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Results: Fixing Parkinson's Disease Motor Behaviours

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6-OHDA mice

Ipsilateral rotation (amphetamine induced)

Wildtype (WT) mice show no rotation

AAV-shPTB: 3 months after injection there was reduction in rotation back to WT levels

AAV-empty: ipsilateral rotation remained at lesioned levels

Contralateral rotation (apomorphine induced)

WT mice show no rotation

AAV-shPTB: 3 months after injection there was reduction in rotation back to WT levels

AAV-empty: contralateral rotation remained at lesioned levels

Limb-use bias test

Parkinson's unilaterally lesioned mice will prefer use of limbs ipsilateral to lesion

WT use both limbs as functionality on both sides

AAV-shPTB: progressive increase in use of contralateral limb (progressive lesioned side recovery)

AAV-empty: touch remained mostly ipsilateral (80% ipsilateral touch)

Silencing of PTB gene (AAV-shPTB) was able to reverse Parkinson's behavioural phenotype within 3 months

Image from Qian et al. (2020)- Figure 6a-c

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Results: Dopamine level and signal restoration in vivo

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Results: Dopamine level and signal restoration in vivo

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High performance liquid chromatography (HPLC) to read dopamine levels

Electrodes measured dopamine release activity- test DA neuron functionality

6-OHDA mice

AAV-shPTB

sharper increase in dopamine levels (65% of original level- dopamine restoration)

dopamine release upon stimulation was detected in both lesioned and intact sides (signal restoration)

AVV-empty

small dopamine level increase

no signal restoration in lesioned side

Image from Qian et al. (2020) - Figure 5 b-e, g, j

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Results: Inducing and restoring DA neurons in vivo

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Results: Inducing and restoring DA neurons in vivo

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comparison of AAV-empty with AAV-shPTB in 6-OHDA mice

AAV-shPTB

10-12 weeks: RFP+TH+ cell bodies and RFP+TH+ fibers in striatum (new DA neurons (TH+) through AAV administration (RFP+))

both had restoration to 30% of intact brain levels

RFP-TH+ fiber increase  (original (RFP-) DA neuron (TH+) axon growth)

AAV-empty

no marked increase in cell bodies or fibers

Image from Qian et al. (2020)- Figure 4b-g

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Lentivirus

small hairpin RNA (shRNA) expressed in lentivirus

transduced into mouse astrocyte to silence gene Ptbp1 (shPTB)

for control, shRNA was not expressed in the virus vector (shCtrl)

used for in vitro testing

Adeno-associated virus (AAV)

shPTB expressed in AAV

Red Fluroescent protein (RFP) attached for tracing

Activated by Cre

compared to empty AAV

RFP replaced with hM4Di (inhibitory muscarinic receptor) in shPTB vector for chemogenetic analysis

hM4Di activated by CNO (clozapine-N-oxide)

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The Gfap-Cre Mouse

Cre-recombinase protein expressed in astrocytes

Use in ASO experiments

Crossed with Rosa-tdTomato mice

Cre protein would transcribe tdTomato marker (red) when activated

ASOs used : PTB-ASO and GFP-ASO (control)

PTB-ASO had 3' fluorescin

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The Induced Parkinson's Disease Model

6-hydroxydopamine (6-OHDA) injected into medial forebrain bundle in Gfap-cre mice

Image created on BioRender.com

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Previous Research - Antisense Oligonucleotides 

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Previous Research - Antisense Oligonucleotides 

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What are Antisense Oligonucleotides?

Antisense Oligonucleotides (ASOs) are short, synthetic, single-stranded oligodeoxynucleotides that can alter RNA and reduce, restore, or modify protein expression through several distinct mechanisms³. One of these mechanisms involves ASOs binding to a specific target mRNA which results in gene silencing.

Research into using ASOs as a therapeutic technique has been growing, following the production of successful ASO therapies such as Spinraza³ which treats spinal muscular atrophy (SMA).

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Impacts on the Field of Transdifferentiation

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Impacts on the Field of Transdifferentiation

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The authors successfully reconstituted a neural pathway using transdifferentiated neurons, a novel discovery in this field⁶.

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Previous Research - Transdifferentiation 

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Previous Research - Transdifferentiation 

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What is Transdifferentiation?
Trandifferentiaion or reprogramming, is a process in which one mature somatic cell is transformed into another mature somatic cell without undergoing an intermediate pluripotent state.

Previous research has highlighted that both mouse and human fibroblasts can be reprogrammed to functional neurons by sequential depletion of the RNA binding proteins PTB and nPTB.

Polypyrimidine tract-binding protein (PTB) suppresses a neuronal induction loop in which microRNA 124 inhibits the transcriptional repressor REST which suppresses many neuronal genes.

Downregulation of PTB induces expression of nPTB (neuronal analog of PTB), which suppresses the transcription activator BRN2 and microRNA 9, both of which are required for neuronal maturation.

Essentially, the downregulation of both PTB and nPTB modulate these loops allowing for neuronal induction and maturation in fibroblasts.

The authors of this paper proposed that PTB knockdown alone could convert astrocytes to neurons, due to the inherent similarities in the nPTB loop between neurons and astrocytes, as shown in the Figure (blue box).

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The authors’ discovery brings great promise for a similar treatment in Parkinson’s Disease. Future studies to research targeting of ASOs to specific neuronal populations, side effects of local astrocyte depletion, as well as overcoming the barrier of age related decreases in neuronal reprogramming ability would all lead the field one step closer to a clinically relevant therapy for patients with Parkinson’s.

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To investigate if transdifferentiation of astrocytes to neurons through PTB knockdown has the potential to restore dopamine, reconstruct neuronal circuits and reverse Parkinson’s disease motor phenotypes in a mouse model.

This paper also investigates how antisense oligonucleotides against PTBP1 (gene that encodes for PTB) could be a potential treatment for PD and other neurodegenerative disorders.

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A short video explaining some common symptoms and possible causes of PD.

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Data availability

RNA-seq data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE142250. Independently generated data are available upon request. Methods have been converted into stepwise protocols and deposited in Protocol Exchange (doi: 10.21203/rs.3.pex-902/v1). Repeats of individual experiments are summarized in Supplementary Table 2, which has been independently verified. All data generated or analysed in this study are included in this published article (and its Supplementary Information files).

Change history

29 July 2020

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

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Nott, A. et al. Brain cell type-specific enhancer-promoter interactome maps and disease-risk association. Science 366, 1134-1139 (2019).

2. 30.
Zhou, H. et al. Glia-to-neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice. Cell 181, 590-603 (2020).

3. 52.
Cohen, J. Statistical Power Analysis for the Behavioral Sciences (Academic Press, 1988).

Download references

Acknowledgements

We thank members of the Fu laboratory for cooperation, reagent sharing and insightful discussion during the course of this investigation and A. Muotri for the gift of the human embryonic stem cell-derived neural progenitors. D.W.C. received a salary from the Ludwig Institute for Cancer Research and is a Nomis Foundation Distinguished Scientist. Z.Z. and X.K. were supported by NSFC grants (31930061, 31761133016, 21790394 and 81974203). W.C.M. and X.-D.F. were supported by a grant from the Larry Hillblom Foundation (2019-A-006-NET). This work was supported by NIH grants (GM049369 and GM052872) to X.-D.F.

Author information

Author notes

1. Jing Hu

Present address: Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People's Hospital, University of Electronic Science and Technology of China, Chengdu, China

2. Yuanchao Xue

Present address: Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

Affiliations

1. Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA, USA

Hao Qian, Jing Hu, Zhengyu Liang, Fan Meng, Xuan Zhang, Yuanchao Xue, Roy Maimon, Steven F. Dowdy, Don W. Cleveland & Xiang-Dong Fu

2. State Key Laboratory of Membrane Biology and Peking-Tsinghua Center for Life Sciences, Institute of Molecular Medicine, Peking University, Beijing, China

Xinjiang Kang & Zhuan Zhou

3. MOE Key Lab of Medical Electrophysiology, ICR, Southwest Medical University, Luzhou, China

Xinjiang Kang

4. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA

Dongyang Zhang & Neal K. Devaraj

5. Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, CA, USA

Roy Maimon & Don W. Cleveland

6. Department of Neurosciences and Center for Neural Circuits and Behavior, University of California, San Diego, La Jolla, CA, USA

William C. Mobley

7. Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA, USA

Xiang-Dong Fu

Contributions

H.Q. and X.-D.F. designed the study. H.Q. performed astrocyte isolation, stereotaxic injection, immunocytochemistry, electrophysiological measurements and behaviour tests. J.H., Y.X. and F.M. contributed to AAV vector construction, immunoblotting and immunocytochemistry and independently characterized astrocyte conversion in vitro. F.M. also contributed to the performance of behaviour tests. Z.L. and F.M. performed RNA-seq and data analysis. H.Q., X.Z., D.Z. and N.K.D. measured striatal dopamine levels. X.K. and Z.Z. recorded activity-induced dopamine release in live animals and on brain slices. S.F.D. supervised ASO design and testing. W.C.M. contributed to analysis and interpretation of neurological data. D.W.C. oversaw biochemical and immunocytochemistry experiments. R.M. independently showed that ASO-mediated suppression of PTB generated new neurons in wild-type mice. R.M. also checked all raw data and verified biological repeats of individual experiments. H.Q., S.F.D., W.C.M., D.W.C. and X.-D.F. wrote the paper.

Corresponding author

Correspondence to Xiang-Dong Fu.

Ethics declarations

Competing interests

X.-D.F. is a founder of CurePharma. The University of California, San Diego has filed a patent application on neuronal reprogramming induced by inactivating PTB by any means for treatment of neurological disorders.

Additional information

Peer review information Nature thanks Ernest Arenas, Anders Bjorklund, Aaron D. Gitler, Malin Parmar and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Cite this article

Qian, H., Kang, X., Hu, J. et al. Reversing a model of Parkinson's disease with in situ converted nigral neurons. Nature 582, 550-556 (2020). https://doi.org/10.1038/s41586-020-2388-4
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Reversing a model of Parkinson's disease with in situ converted nigral neurons

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Parkinson's disease is characterized by loss of dopamine neurons in the substantia nigra1. Similar to other major neurodegenerative disorders, there are no disease-modifying treatments for Parkinson's disease. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits2.

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What is Parkinson's Disease?

Parkinson’s Disease (PD) is a common neurodegenerative disorder, affecting 2-3% of the population aged 65+².

PD is characterised by the loss of dopaminergic (DA) neurons in the substantia nigra, which causes striatal dopamine deficiency and diminished dopamine concentrations in the nigrostriatal pathway. The main function of this pathway is voluntary movement.

As this disease progresses, the loss of neurons becomes more widespread, and the disruption of neuronal circuits results in a number of motor symptoms such as bradykinesia and tremors².

Similarly to other major neurodegenerative disorders, there is no cure for Parkinson’s Disease. However, a proposed potential treatment is to replace lost neurons to reconstruct neuronal circuits, through in vivo transdifferentiation.

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Effective controls, careful cell lineage tracking

Motor effects attributed to neuron conversion

Testing verified in vitro as well as in vivo

Age-matched to onset

ASOs are clinically proven to remedy neurological diseases³

Strengths of this paper include: detailed tracking of cell type and origin by monitoring the expression of numerous markers (as explained in methods), examining both the molecular mechanism of astrocyte conversion (the PTB pathway) as well as the rescuing effects on the disease model and disease related motor phenotypes (a clinically relevant application). As well, the authors verified results both in vivo and in vitro, removing any discrepancies that one method may have procured over the other. Another major strength of the paper is that the authors tested 2 month old mice and 1 year old mice. This is an age comparable in humans to the age of onset of Parkinson’s. This allows the authors to discern age-related effects/deficiencies on neuronal reprogramming. ASOs are able to diffuse t/membranes, do not have immunity response, have been used in treatment in other neurological diseases such as spinal muscular atrophy (Spinraza)². Furthermore, clinical tests confirm the safety of this method.

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Independent Research Reveals Similar PTB Insights

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Independent Research Reveals Similar PTB Insights

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PTB knockdown was used to induce astrocyte to DA neurons in the SN to similarly ameliorate Parkinson’s symptoms in Zhou et al 2020⁴. This supports the authors' findings in this paper.

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Highlights

*

Knockdown of Ptbp1 converts Müller glia into retinal ganglion cells in mature retinas

*

Central projections of converted retinal ganglion cells restore visual responses

*

Induction of neurons with dopaminergic features in PD model mice

*

Induced neurons alleviated motor dysfunctions in PD mice

Summary

Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury. Previous attempts in this area using expression of transcription factors were hindered by the low conversion efficiency and failure of generating desired neuronal types in vivo. Here, we report that downregulation of a single RNA-binding protein, polypyrimidine tract-binding protein 1 ( Ptbp1), using in vivo viral delivery of a recently developed RNA-targeting CRISPR system CasRx, resulted in the conversion of Müller glia into retinal ganglion cells (RGCs) with a high efficiency, leading to the alleviation of disease symptoms associated with RGC loss. Furthermore, this approach also induced neurons with dopaminergic features in the striatum and alleviated motor defects in a Parkinson's disease mouse model. Thus, glia-to-neuron conversion by CasRx-mediated Ptbp1 knockdown represents a promising in vivo genetic approach for treating a variety of disorders due to neuronal loss.

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Glia-to-Neuron Conversion by CRISPR-CasRx Alleviates Symptoms of Neurological Disease in Mice

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Knockdown of Ptbp1 converts Müller glia into retinal ganglion cells in mature retinas * Central projections of converted retinal ganglion cells restore visual responses * Induction of neurons with dopaminergic features in PD model mice * Induced neurons alleviated motor dysfunctions in PD mice Conversion of glial cells into functional neurons represents a potential therapeutic approach for replenishing neuronal loss associated with neurodegenerative diseases and brain injury.

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6-OHDA lesion mice fail to exhibit a number of disease phenotypes

No good alternative, since while genetic methods recapitulate alpha-synuclein pathology, DA neuron degeneration is missing⁵

A major weakness of this paper is the incomplete recapitulation of disease phenotypes in the mouse model used by the authors. This includes the lack of Lewy body formation, bradykinesia and dementia. The lack of such phenotypes is important since they are major components of the disease and necessarily implies that there are molecular interactions seen in the disease that are unaccounted for in this study³. Essentially authors are held back by the lack of an optimal disease model. Dementia comorbid with Parkinson’s is known to have alpha-syn and tau pathologies, an aspect not taken into account in this study. Nevertheless, this model was the best option for the author’s means since it does recapitulate the degradation of the nigrostriatal dopamine pathway, while other models (i.e. genetic) are not consistent in that respect. Beal 2010

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Were the goals of the project met?

The authors successfully demonstrated the direct conversion of astrocytes to dopaminergic neurons of an in vitro mouse model of Parkinson. These newly-created neurons were shown to integrate into the dopamine pathway, functionally replacing lost neurons in the disease model. The authors were also able to recreate the same effects using an antisense oligonucleotide targeting PTB.

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1. Qian, H., Kang, X., Hu, J., Zhang, D., Liang, Z., Meng, F., Zhang, X., Xue, Y., Maimon, R., Dowdy, S.F., et al. (2020). Reversing a model of Parkinson’s disease with in situ converted nigral neurons. Nature 582, 550–556.

2. Poewe W. et al. Parkinson's disease. Nat Rev Dis Primers 3, 1-21 (2017).

3. Finkel, R.S.; Mercuri, E.; Darras, B.T.; Connolly, A.M.; Kuntz, N.L.; Kirschner, J.; Chiriboga, C.A.; Saito, K.; Servais, L.; Tizzano, E.; et al. Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy. N. Engl. J. Med 377, 1723–1732 (2017).

4. Zhou, H. et al. Glia-to-neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice. Cell 181, 590-603 (2020).

5. Beal, M. Parkinson's disease: a model dilemma. Nature 466, S8–S10 (2010). https://doi.org/10.1038/466S8a

6.  Gascón, S., Masserdotti, G., Russo, G. L. & Götz, M. Direct Neuronal Reprogramming: Achievements, Hurdles, and New Roads to Success. Cell Stem Cell 21, 18–34 (2017).

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Introduction

Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the nigrostriatal pathway1. Clinical manifestations of PD include bradykinesia, muscular rigidity, resting tremors, and postural instability. Although replacement therapy with the dopamine precursor, levodopa, is effective for these symptoms of PD2, chronic treatment is associated with the development of motor complications and the disease is inexorably progressive3,4-possibly because degeneration of nigrostriatal neurons causes progressive dopamine loss.

α-synuclein (SNCA) protein is a major component of the Lewy body, a pathological hallmark of both the sporadic and familial forms of PD5,6, which suggests that dysfunction or toxicity caused by SNCA protein results in the pathology of PD. A missense mutation in the SNCA gene was first identified in PARK1, a dominant familial form of PD7. In addition, duplication or triplication of the SNCA gene has been shown to cause the dominant form of PD, PARK48,9. Polymorphisms in the SNCA gene are also associated with susceptibility to sporadic PD and tend to be correlated with SNCA mRNA levels10. Our group and other researchers have shown that wild type SNCA causes cell-autonomous toxicity when expressed specifically in nigral dopaminergic neurons11,12,13,14. Because SNCA pathology extends from the spinal cord, brainstem, or olfactory bulb to the cortex during PD progression15,16,17,18, decreasing SNCA expression levels could be an attractive treatment for suppressing PD.

The antisense oligonucleotide (ASO) is a potential gene therapy for targeting the SNCA gene. ASO-based therapies have already been approved for neuromuscular diseases including spinal muscular atrophy (SMA) and Duchenne muscular dystrophy19,20,21,22.

The nucleic acids of ASO can be modified to acquire high nuclease resistance and efficient binding affinities toward complementary strands23,24. We have shown that ASOs with amido-bridged nucleic acid (AmNA), an analog of locked nucleic acid (LNA) with modification of the amide bond bridged between the 2′ and 4′ carbons of the ribose, show higher knockdown efficiency and safety compared to natural ASO and LNA25,26,27 (Supplemental Fig. 1a).

In the present study, our designed AmNA-ASO significantly reduced human SNCA (hSNCA) mRNA and protein levels in human cultured cells and in mice. After administering AmNA-ASO into the intracerebroventricular space of the mouse brain, AmNA-ASO was widely distributed throughout the brain and efficiently taken up by neuronal and, to a lesser extent, non-neuronal cells without the aid of additional chemicals. Moreover, a single injection of AmNA-ASO ameliorated the defects observed in transgenic mice expressing wild type SNCA. Hence, this work highlights the potential of SNCA-targeted AmNA-ASO therapy for PD.

Results

AmNA-ASO efficiently reduced SNCA mRNA levels in human cultured cells

To determine the potency of AmNA-ASO for reducing the levels of SNCA mRNA, we generated a series of AmNA-ASOs (n = 50) covering 80.7% of the coding sequence of SNCA mRNA. The AmNA-ASOs were designed to be 15-mer chimeric antisense oligonucleotides (gapmer) containing AmNA. Supplemental Fig. 1b illustrates the designed AmNA-ASO, which contains AmNA at each end flanking the central bases of DNA with a gapmer motif of 3AmNA-9DNA-2AmNA-1DNA (3-9-2-1).

We screened synthesized AmNA-ASOs for knockdown efficiency of SNCA mRNA in human embryonic kidney 293 (HEK293) cells that express hSNCA mRNA endogenously. For screening, we transfected AmNA-ASOs into HEK293 cells at a single concentration (50 nM) and quantified the SNCA mRNA level using quantitative polymerase chain reaction (qPCR) 24 hours after transfection. We found that several AmNA-ASOs significantly reduced SNCA mRNA levels. In particular, AmNA-ASO No.19 significantly decreased the SNCA mRNA level to 24.5% of the normal expression level in mock transfected cells (control = 100% ± 8.52%, AmNA-ASO No.19 = 24.5% ± 2.29%, p < 0.01 by Dunnett's test), suggesting that AmNA-ASO No. 19 is highly potent for targeting SNCA mRNA in human cultured cells (Fig. 1a). To determine the most efficient construct of AmNA-ASO No. 19, we generated AmNA-ASOs No. 19 with variable lengths and gapmer motif modifications. As a control, we used phosphate-buffered saline (PBS) and scr-AmNA containing the same base composition as AmNA-ASOs No. 19 but in a scrambled order (Fig. 1b). We then transfected the ASOs into HEK293 cells and measured the level of SNCA mRNA expression by qPCR 24 hours after transfection. We found that all of the tested AmNA-ASOs significantly reduced the SNCA mRNA level. AmNA-ASO No. 19 with the gapmer motif of 3AmNA-9DNA-2AmNA-1DNA (3-9-2-1), the same sequence used for the screening described above, was the most efficient and downregulated the SNCA mRNA level to 19.0%, suggesting that AmNA-ASO No. 19 3-9-2-1 (hereinafter referred to as ASO A19) is highly potent for targeting SNCA mRNA in the human cultured cells (Fig. 1c).

ASO A19 (3-9-2-1) efficiently downregulated the level of SNCA mRNA in HEK293 cells. ( a) qPCR analysis showing the expression levels of SNCA mRNA in HEK293 cells. A total of 50 AmNA-ASOs were individually transfected into HEK293 cells. ASO A19 significantly reduced levels of hSNCA mRNA. Data are expressed as mean ± SEM (n = 6). **p < 0.01 by Dunnett's test. ( b) Sequences of ASO A19 containing various gapmer motifs. Capital and small letters represent AmNA and DNA, respectively. ( c) Assessment of ASO A19 with variable gapmer motifs. qPCR analysis showing the expression levels of SNCA mRNA in HEK293 cells. ASO A19 (3-9-2-1) reduced SNCA mRNA more efficiently than the other modified ASO A19. Data are expressed as mean ± SEM (n = 3).

AmNA-ASOs were shown to be less toxic to animals than LNA-ASOs27. To compare the knockdown efficiencies of AmNA- and LNA-ASOs, we generated LNA-ASO No. 19 3-9-2-1 (hereinafter referred to as ASO L19) carrying the same target sequence as ASO A19. We transfected ASO A19 and ASO L19 into HEK293 cells and measured the levels of SNCA mRNA expression by qPCR 24, 72, and 96 hours after transfection, as described above. We found that ASO A19 reduced the SNCA mRNA levels to 10.3% (24 h) and 15.2% (72 h), whereas ASO L19 reduced the levels to 18.3% (24 h) and 28.1% (72 h), suggesting that ASO modified with AmNA knocked down SNCA mRNA more efficiently than that modified with LNA (p = 0.0076, one-way ANOVA, Tukey post hoc test) (Fig. 2a). To evaluate the off-target effects of ASO A19, we measured the levels of β-synuclein (SNCB) and γ-synuclein (SNCG), which are members of the synuclein family. We found no significant difference in mRNA levels between control and ASO A19-treated cells, suggesting no off-target effects of ASO A19 on SNCB and SNCG (Fig. 2b). We also evaluated SNCA protein levels by Western blotting 24, 48, 72, and 96 hours after transfection. ASO A19 reduced the expression levels of SNCA protein to similar levels as ASO L19 at both 72 and 96 hours after transfection (Fig. 2c,d and Supplemental Fig. 2), although protein levels were not yet reduced at 24 and 48 hours (Supplemental Fig. 3). Taken together, these data indicate that ASO A19 can reduce SNCA mRNA and protein levels in human cultured cells and that the potency of ASO A19 is comparable to or more efficient than that of ASO L19.

ASO A19 was more efficient than ASO L19 in HEK293 cells. ( a) qPCR analysis showing SNCA mRNA in HEK293 cells transfected with ASO A19 and ASO L19. ASO A19 decreased the expression levels of SNCA mRNA compared to ASO L19 24 and 72 hours after transfection. Data are expressed as mean ± SEM (n = 3). *p < 0.01 by one-way ANOVA, Tukey post hoc test. ( b) qPCR analysis of SNCA, SNCB, and SNCG mRNA in HEK293 cells transfected with ASO A19 and ASO L19. Although ASO A19 decreased the expression levels of SNCA mRNA (see a for comparison), SNCB and SNCG levels showed no reduction. *p < 0.01 by one-way ANOVA, Tukey post hoc test. ( c, d) Immunoblot and densitometry analysis showing that both ASO A19 and ASO L19 reduced SNCA protein levels. Proteins were extracted from HEK293 cells 72 and 96 hours after transfection of ASO A19 and ASO L19 and subjected to immunobloting. Images of blots were cropped from different parts of the same gel. Full-length gels are shown in Supplemental Fig. 2. Immunoblotting with anti-beta-actin was used as an internal control. Densitometry data are expressed as mean ± SEM (n = 2). ( e) Alignment and relationship of the base sequences of four: synuclein family, human SNCA, SNCB, SNCG, and mouse Snca. The square enclosed area represents the sequence that ASO A19 can target.

ASOA19 was efficiently delivered into mouse brains, unassisted by transfection reagents

To establish a future ASO treatment for PD, we determined if ASO A19 was effective for targeting SNCA in PD model mice. Intrathecal administration of ASO has recently been established for the treatment of patients affected with SMA20. To determine if ASO A19 can be delivered from cerebrospinal fluid (CSF) space to neurons in the central nervous system (CNS), we injected ASO A19 conjugated with fluorescent Alexa 488 into the left lateral ventricle of mice. We extracted the mouse brains 48 hours after injection and examined the localization of ASO. Auto-fluorescence images showed that ASO A19 conjugated with Alexa 488 was widely distributed in the CNS after intracerebroventricular injection (Fig. 3a). Immunofluorescence images showed that ASO A19 was localized in tyrosine hydroxylase (TH) positive neurons in the substantia nigra (arrow in Fig. 3c-c") and neurons surrounded by TH-positive axons in the striatum (arrow in Fig. 3e-e"), suggesting that ASO A19 could be efficiently delivered to TH- and non-TH-positive neurons in vivo (Fig. 3b-e). To further characterize the cell types that take up ASO A19, we immunostained the mouse brain sections for NeuN and MAP2 (neuronal markers), GFAP (astrocytes marker), O4 (oligodendrocytes marker), and Iba1 (microglial marker). ASO A19 was mainly taken up by neuronal cells (arrow in Fig. 4a,b) and, to some extent, by oligodendrocytes, astrocytes, and microglial cells (arrow in Fig. 4c-e). Furthermore, we evaluated the brain distribution of ASO A19 and found ASO A19 in various brain areas including cortex, olfactory bulb, hippocampus, dentate gyrus, striatum, substantia nigra, cerebellum, and brain stem (Fig. 5). These results suggest that ASO injected into the left ventricle is distributed into various cell types and brain areas without the aid of chemical reagents.

ASO A19 injected into the lateral ventricle was efficiently delivered throughout the mouse brain. ( a) Auto-fluorescence image of the brain showing efficient delivery of ASO A19 by intracerebroventricular injection. Fluorophore-conjugated ASO A19 was widely distributed into brain 48 hours after injection into the left lateral ventricle. A non-injected brain (left) and brain injected with ASO A19 conjugated with Alexa 488 (right) are shown. ( b- e) Uptake of ASO A19 by presynaptic and postsynaptic tyrosine hydroxylase (TH)-positive neurons. ASO A19 was localized in TH-positive neurons in the substantia nigra (SN) and distributed into neurons contacting TH immunoreactive axons in the striatum (Str) (arrow). Immunofluorescence images of the SN (b and c) and striatum ( d, e). Auto-fluorescence of AmNA-Alexa488 ( b- e) and immunofluorescence with TH ( b'- e'). Merged images showing AmNA-Alexa488 (in green), TH (in red), and DAPI-labeled cell nuclei (in blue) ( b"- e").

Uptake of ASO A19 by various cell types in the mouse brain. Immunostaining images of the ( a) substantia nigra and ( b- e) striatum of Alexa 488 conjugated-ASO A19-administered wild type mice. Uptake of ASO A19 by neurons, astrocytes, oligodendrocytes, and microglia was seen. ASO A19 was distributed into cells stained with NeuN ( a), MAP2 ( b), GFAP ( c), O4 ( d), and Iba1 ( e) (arrow). Merged images showing AmNA-Alexa488 in green, NeuN ( a), MAP2 ( b), GFAP ( c), O4 ( d), Iba1 ( e) in red, and DAPI-labeled cell nuclei in blue. Scale bar: 20 μm.

Widespread distribution of ASO A19 throughout the mouse brain. Immunostaining images of brain sections collected from Alexa 488 conjugated-ASO A19-administered wild type mice. Widespread distribution of ASO A19 in different brain areas, including the cortex, olfactory bulb, hippocampus, dentate gyrus, striatum, substantia nigra, cerebellum, and brain stem, was seen. Merged images showing AmNA-Alexa488 in green and DAPI-labeled cell nuclei in blue. Scale bar: 20 μm.

ASOA19 efficiently targeted SNCA in transgenic mice expressing hSNCA

To determine if ASO A19 injected into the ventricle could reduce hSNCA expression in the CNS, we tested its effects in the transgenic PD mouse model TH-SNCA-140 m. TH-SNCA-140 m mice carry the hSNCA gene with an Ala53Thr mutation, the expression of which is driven by the TH promoter28. We injected 100 μg of control ASO (ASO containing scrambled sequences) and ASO A19 into the left lateral ventricles of two-month-old TH-SNCA-140 m transgenic mice in a single application. Neither injection caused any toxicity-related changes in general health status or behavior. Two weeks after injection, we extracted mRNA from the left cerebral hemisphere and measured hSNCA mRNA levels. Quantification by qPCR showed that ASO A19 significantly decreased hSNCA mRNA levels relative to control ASO (100% (control) vs. 47.2% (ASO A19), p < 0.05, t-test, n = 5), which suggests that ASO A19 injected into the CSF can reduce hSNCA mRNA levels in the brain (Fig. 6a). To evaluate the off-target effects of ASO A19, we measured the levels of endogenous α-synuclein (Snca), β-synuclein (Sncb), and γ-synuclein (Sncg) in mice. We found no significant differences in mRNA levels of these substances between control and ASO A19-treated mice, suggesting that ASO A19 has no off-target effects (Fig. 6b).

Intracerebroventricular administration of ASO A19 reduced SNCA mRNA and protein levels in brains of TH-SNCA mice. ( a, b) qPCR analysis of mRNA levels of hSNCA and mouse endogenous Snca, Sncb, and Sncg in mouse brains. ASO A19 or control was injected into the left lateral ventricle of TH-SNCA-140 m mice expressing human SNCA with Ala53Thr mutation. Total RNA was extracted from left cerebral hemisphere samples collected 14 days after ASO A19 injection. ASO A19 significantly reduced the levels of SNCA mRNA. The levels of Snca, Sncb, and Sncg mRNA showed no reduction. Data are expressed as mean ± SEM (n = 5). *p < 0.05 by t-test. ( c, d) Immunoblotting and densitometry analysis showing that ASO A19 reduced the levels of SNCA protein. Proteins were extracted from right cerebral hemisphere samples two weeks after administration of ASO A19 and subjected to immunoblotting. The images of blots were cropped from different parts of the same gel. Full-length gels are shown in Supplemental Fig. 4. Immunoblotting with anti-beta-actin was used as an internal control. ASO A19 significantly reduced the levels of SNCA protein. Densitometry data are expressed as mean ± SEM (n = 5).

We also evaluated hSNCA protein levels in the right cerebral hemisphere by Western blotting two weeks after injection. ASO A19 significantly reduced the expression levels of SNCA protein (Fig. 6c,d and Supplemental Fig. 4). Taken together, these data indicate that ASO A19 can reduce SNCA mRNA and protein levels in the brains of TH-SNCA-140 m transgenic mice.

AmNA-ASO ameliorated defects in motor behavior observed in hSNCA transgenic mice

To further confirm the efficacy of ASO A19, we tested it in another transgenic mouse line Thy1-SNCA, which carries human wild type SNCA driven by the murine thymus cell antigen 1 (Thy1) promoter. High levels of hSNCA expression are present in the neocortex, hippocampus, olfactory bulb, thalamus, colliculi, substantia nigra, and brainstem of Thy1-SNCA mice29. Moreover, Thy1-SNCA mice have a key feature of PD: high levels of SNCA phosphorylated at serine 129, which is shown to be the predominant form of SNCA found in Lewy bodies, in the substantia nigra, striatum, cortex, frontal cortex, and hippocampus30.

In this mouse model, we quantified expression levels of hSNCA protein extracted from left cerebral hemisphere by enzyme-linked immunosorbent assay (ELISA), which is quantitatively sensitive to detect changes in the level of highly expressed hSNCA protein by the Thy1 promoter. We found that hSNCA protein appeared to partition primarily into the Triton X-100 soluble fraction, in contrast to the 10-fold smaller fraction of hSNCA protein in the insoluble fraction. ELISA showed that the levels of SNCA protein in ASO A19-treated mice were significantly decreased in both the soluble and insoluble fractions (soluble hSNCA protein in ASO-injected mice/control = 87.3% ± 2.9%, p = 0.006, t-test and insoluble hSNCA protein in ASO-injected mice/control = 81.5% ± 3.6%, p = 0.0073, t-test), indicating that ASO A19 efficiently reduced hSNCA protein levels in the brains of Thy-1 SNCA mice (Supplemental Fig. 5). Histological evaluation of brain sections from wild type, PBS-, and ASO A19-injected Thy-1 SNCA mice four weeks after treatment with an antibody specific to hSNCA further confirmed SNCA protein knockdown in ASO A19-injected mice of the 3 groups (Supplemental Fig. 6). In addition, to assess the effect of SNCA knockdown on the formation of inclusions, we immunostained the brain sections for phosphorylated SNCA, which is frequently used as an indicator for SNCA inclusions. Partial decrease of phosphorylated SNCA density was seen in the cortex, dentate gyrus, and olfactory bulb of ASO A19-injected Thy-1 SNCA mice (Fig. 7).

Decrease of phosphorylated SNCA in Thy1-SNCA mouse brains after ASO A19 treatment. Immunohistological images of various brain areas in wild type, control, and ASO A19-treated Thy-1 SNCA mice using a monoclonal antibody that recognizes phosphorylated SNCA. ASO A19-treated Thy1-SNCA mice showed partial reduction of phosphorylated SNCA in the cortex, olfactory bulb, and dentate gyrus relative to control mice (arrow). Scale bars: 100 μm.

Thy-1 SNCA mice also show significant impairments in motor performance at the age of two months, which can be reversed by the dopamine precursor L-dopa, similar to PARK4 and other PD patients29,30,31. To determine if ASO A19 could ameliorate behavioral defects in Thy1-SNCA mice, we examined motor behavior in control and ASO A19-injected mice using the wire suspension test every seven days after injection (See Material and Method). We found that mice injected with ASO A19 showed better performance than control Thy1-SNCA mice. The mean test time for ASO A19-injected mice was 67.1 s on day 27 after injection, whereas the mean time was 48.2 s for control mice (Fig. 8a). In addition, mice injected with ASO A19 tended to show improvement in the beam walk test, which was performed 28 days after injection using two beams with different widths. We measured the frequency of slips from the beam and normalized this value to the walking speed along the beams. ASO A19-injected mice showed tendency of reduced frequency of slips per speed compared to controls (ASO A19 vs. control: 168.7 vs 378.8 per m/s (beam 1) and 248.8 vs. 438.1 per m/s (beam 2), Fig. 8b). Lastly, we examined the effects of ASO A19 on the pasta gnawing test, which can sensitively evaluate eating behavior that requires fine coordination of limb and oromotor functions32,33,34. Notably, ASO A19-injected mice showed significantly better performances than control mice. On day 20 after injection, the mean number of bites per episode was 3.94 for control mice and 6.44 for ASO A19-injected mice (p = 0.0051, t-test). On day 27 after injection, the mean number of bites per episode was 3.92 for control mice and 6.8 for ASO A19-injected mice (p = 0.0021, t-test) (Fig. 8c). Taken together, these data indicate that ASO A19 can improve behavioral defects observed in Thy1-SNCA mice accompanied by downregulation of SNCA.

Intracerebroventricular administration of ASO A19 improved motor function in Thy1-SNCA mice. ( a) Hanging time for the wire suspension test, which was performed every seven days after treatment with ASO A19 or control. ASO A19-treated mice showed a trend of longer suspension compared with controls. Data are expressed as mean ± SEM (n = 17 ASO A19 and 13 control). ( b) The beam walking test was performed four weeks after administration of ASO A19 or control. The graph shows slips per speed. ASO A19-treated mice showed a trend of fewer slips than controls. Data are expressed as mean ± SEM (n = 17 ASO A19 and 13 control). ( c) Pasta gnawing test showing the number of bites per gnawing episode every seven days after administration of ASO A19 or control. ASO A19-treated mice showed an increased number of bites, indicating improvement in hypokinetic defects. Data are expressed as mean ± SEM (n = 17 ASO A19 and 13 control). **p < 0.01 by t-test.

Discussion

In the present study, we generated ASO A19 capable of reducing the level of hSNCA mRNA in HEK293 cells and found that ASO A19 is superior to ASO L19 for decreasing hSNCA mRNA. Further, we confirmed that ASO A19 was widely distributed in the CNS following a single intracerebroventricular administration. This study is the first to report that intracerebroventricular administration of ASO in the absence of any carrier or conjugation can knockdown the level of hSNCA and improve motor dysfunction in transgenic animal PD models.

SNCA protein is one of the most promising therapeutic targets for the treatment of PD35. We showed that ASO A ¹⁹ can target wild type and mutant hSNCA, indicating its applicability for both familial and sporadic PD. Moreover, reducing the level of SNCA expression is likely to protect against neuronal toxicities. We previously reported that reduction of SNCA expression enabled the survival of more TH-positive neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model rats36. SNCA knockdown by shRNA attenuates the progression of motor deficits in rotenone-exposed rats37, suggesting that reduction of SNCA expression might also be beneficial for PD. Benskey et al. reported that excessive reduction of SNCA within nigrostriatal neurons induced rapid up-regulation of the major histocompatibility complex class 1 (MHC-1) and resulted in the death of affected neurons38. However, several studies have shown that normalization of overexpressed SNCA decreased neurodegeneration of dopaminergic neurons and improved motor function in animal models37,39. These data imply that adequate downregulation of SNCA can prevent neurodegeneration with little disadvantages.

To improve the efficacy of antisense oligonucleotides for in vivo therapeutic application, we developed a less toxic ASO containing AmNA25,26. We then applied AmNA-ASO targeting Y-box binding protein-1 effectively as an antiangiogenic cancer therapy27. To increase ASO stability in vivo and reduce the number of treatments, we applied the technique of AmNA modification in ASO targeting hSNCA. Importantly, AmNA modification enhanced the potency of ASO for targeting SNCA (Fig. 2a-d). Consistently, single intracerebroventricular administration of ASO A19 was effective for targeting SNCA in the mouse CNS even two weeks after injection (Fig. 6c,d), supporting the stability of intracerebroventricularly administered ASO A19 in patients with α-synucleinopathies.

In an alternative approach to targeting SNCA protein, antibodies against the C-terminus of SNCA protein were shown to reduce accumulation of C-terminal truncated SNCA protein, prevent TH loss in the striatum, and improve motor deficits in SNCA transgenic mouse models40. A phase I study of administration of this anti-SNCA antibody, PRX002, was performed and showed that serum SNCA protein could be modulated without any serious adverse events41. However, this anti-SNCA immunotherapy works primarily by recognizing and clearing extracellular SNCA protein to block cell-to-cell propagation of SNCA in the brain and is less effective at targeting intraneuronal SNCA. ASO A19 administration could be a complementary approach used with anti-SNCA immunotherapy.

Although we found no off-target or toxic effects of ASO A19 (Fig. 6b) (Supplemental Fig. 7), the safety of ASO A19 injection must be confirmed for future clinical applications. Studies using SNCA knock-out mice showed that SNCA might be required for synaptic function42,43 and maintenance of subependymal neural stem cells44. It is important to note that antisense therapy does not cause total loss of SNCA expression (Fig. 6a). Hence, a modest reduction of SNCA expression is not likely to cause serious defects, even if SNCA is required for some biological functions in the human brain.

In conclusion, we demonstrated that intracerebroventricular administration of AmNA-ASO can reduce SNCA mRNA and the corresponding protein levels, resulting in improvement of some motor dysfunctions observed in PD model mice. Remarkably, intracerebroventricular administration of nusinersen, an ASO for treating SMA, was recently approved19,20,21,22. Our findings highlight the potential of AmNA-ASO as a novel therapy for PD and other synucleinopathies.

Material and Methods

Oligonucleotides

The ASOs used in this study, which were synthesized by GeneDesign, are gapmer-type 13-15-mer phosphorothioate oligonucleotides containing five AmNA modifications (Supplemental Fig. 1b).

AmNA-ASOs containing specific DNA sequences (TCC and TGC), which have been shown to bind to hepatocellular proteins and cause hepatotoxicity45, induce cellular responses mediated by Toll-like receptor 9, and cause strong inflammatory responses (CpG), were excluded46. The loop structure of SNCA mRNA, where ASOs easily bind target RNA, was predicted using the mFold web server47 and the 50 most effective sequences targeting hSNCA mRNA were selected.

Cell culture and transfection

HEK293 cells (ATCC CRL-1573) were cultured in Dulbecco's Modified Eagle Medium (Sigma) supplemented with 10% fetal bovine serum at 37 °C in a humidified chamber of 95% O ₂ and 5% CO ₂. Cells were plated into 12-well plates 24 hours before transfection. AmNA-ASOs were transfected into HEK293 cells with Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocols. The final concentration of AmNA-ASOs was adjusted to 50 nM. All ASOs were tested in triplicate.

Quantitative RT-PCR of total RNA extracted from HEK293 cells and mouse brains

Total RNA was extracted from HEK293 cells using a RNeasy Mini kit (QIAGEN). RNA was reverse transcribed to complementary DNA (cDNA) using the Superscript III First Strand cDNA system (Life Technologies) according to the manufacturer's protocol and cDNA was used as the template for qPCR. qPCR was performed using TaqMan Gene Expression assays on an ABI PRISM 7900HT Sequence Detection System (Life Technologies). Relative mRNA expression was normalized by the 18 s ribosomal RNA level in each sample and calculated using the delta-delta Ct method. Taqman primer/probe sets specific for hSNCA, hSNCB, hSNCG, mouse Snca, mouse Sncb, mouse Sncg, and 18 s ribosomal RNA were purchased from Thermo Fisher Scientific.

To quantify hSNCA mRNA levels in mice, brain samples were collected and separated into left and right cerebral hemispheres. Left hemispheres were used for qPCR and right hemispheres for Western blot analysis. Left hemispheres were immediately snap-frozen in liquid nitrogen and stored at −80 °C. For analysis, frozen left brain hemispheres were ground using a prechilled mortar and pestle with liquid nitrogen occasionally added to prevent thawing. After the tissue was finely ground, ISOGEN (Nippon gene) was added to isolate total RNA, according to the manufacturer's instructions. Reverse transcription and qPCR were performed as described above.

Western blot analysis of SNCA protein from HEK293 cells and mouse brains

Proteins were extracted from HEK293 cells using the CelLytic M Cell Lysis Reagent (Sigma-Aldrich) with protease and phosphatase inhibitor cocktail. Protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Cell lysates were mixed with Laemlli sample buffer, boiled, and separated on polyacrylamide gel. After protein transfer to polyvinylidene fluoride membranes, the blots were probed with the primary antibodies, α-synuclein (BD Transduction Laboratories, cat #610787, 1:10000 dilution) and actin (Millipore, cat #MAB1501, 1:1000 dilution); visualized with secondary antibodies, HRP-conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare); and developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare). The signals of immunoblots were acquired with a Chemidoc Touch Imaging System (Bio-Rad).

To quantify hSNCA protein in mouse brains, proteins were extracted from the right cerebral hemispheres using the CelLytic™ MT Cell Lysis Reagent for mammalian tissues (Sigma-Aldrich) with protease and phosphatase inhibitor cocktail. The BCA assay and Western blotting were performed as described above. The primary antibody used for hSNCA detection was α-synuclein (211) (Santa Cruz, cat #sc-12767, 1:1000 dilution).

Mouse strains

TH-SNCA-140 m transgenic mice with a C57BL/6J background carrying the hSNCA gene with a Ala53Thr mutation, for which expression is driven by the TH promoter28, and Thy1-SNCA transgenic mice (Line 61) with a C57BL/6 × DBA/2F1 background carrying human wild type SNCA driven by the murine thymus cell antigen 1 promoter29,30,31 were used.

Animal housing

Mice were housed in individual ventilated cages. Each cage contained a maximum of four mice of the same sex. Mice were housed under a 12 hours light/dark cycle and standard housing conditions with ad libitum access to food and water for one week before the experiment. All animal experiments were conducted according to ARRIVE guidelines.

Intracerebroventricular injection of AmNA-ASOs into mice

Mice were anesthetized with an intraperitoneal injection of cocktails comprising medetomidine (2 mg/kg), midazolam (10 mg/kg), butorphanol (1 mg/kg) or ketamine (50 mg/kg), medetomidine (0.3 mg/kg), and butorphanol (5 mg/kg). Anesthetized mice were placed into a stereotaxic frame and a single burr hole was drilled to allow placement of the stereotactic needle (Hamilton®). The coordinates for injection were 0.3 mm anterior and 1.0 mm lateral to Bregma and 3 mm below the dura. AmNA-ASOs dissolved in 10 μl phosphate-buffered saline (PBS) were injected into the left lateral ventricle at a rate of 2 μl/min48.

Behavioral testing

For mice administered with AmNA-ASOs, body weight was measured and pasta gnawing test32 and wire suspension test49 were performed four times per week. The beam walk test50,51 was performed once after the final pasta gnawing and wire suspension tests. For the pasta gnawing test, mice were brought into the experimental room at least two hours before testing. The cage top, water bottle, and food pellets were removed (only the cage lid remained) and a small piece of dry spaghetti (approximately 5 mm) was placed in the cage. After habituation, the home cage was placed on the testing table. Spaghetti noodles were broken into several pieces approximately 1 cm long and placed in the center of the cage. A microphone was placed above the noodle pieces and recording was initiated as soon as the animal started to eat. The number of bites per gnawing episode and biting frequency were evaluated. The acquired gnawing pattern data were analyzed using Avisoft SASLab Pro.

For the wire suspension test, a wire cage lid was used with duct tape placed around the perimeter to prevent the mouse from walking off the edge. The animal was placed on top of the cage lid and the lid was lightly shaken three times to force the mouse to grip the wires. The lid was then turned upside down and held approximately 50-60 cm above a soft underlay, high enough to prevent the mouse from jumping down but not high enough to cause harm in the event of a fall. The latency to fall was quantified.

For the beam walk test, we used long strips of wood (beams) with a 1000 × 13 or 10 mm cross-section. The beams were placed horizontally 50 cm above the surface, with one end mounted on a narrow support and the other end attached to the home cage into which the mouse could escape. A desk lamp was positioned above the start of the beam. Three training trials were performed prior to testing using three different starting points on the 20 mm square beam: close proximity to the home cage (trial 1), the center of the beam (trial 2), and at the brightly illuminated end of the beam (trial 3). After the mice were trained, they underwent test trials on each of the square beams. The test trials were videotaped and then evaluated with Observer XT 10.5 (Noldus). The latency to traverse each beam and the number of times the hind feet slipped off each beam were recorded for each trial.

Immunofluorescence analysis

Frozen brain hemispheres from non-injected and Alexa 488-conjugated AmNa-injected C57BL/6 mice were cryosectioned coronally at 30 µm thickness on a Leica CM1850. Sections were transferred to 30% sucrose solution in PBS (Thermo Scientific) and stored at 4 °C. Immunofluorescence experiments were performed using the following protocol: sections were washed 3 × 5 min in PBS, fixed with 4% PFA in PBS for 10 min, washed 3 × 5 min in PBS with 0.3% Triton X-100 (PBST), blocked 1 hour with 10% Block Ace PBS, incubated with primary antibodies in 10% Block Ace PBS overnight at 4 °C, washed 3 × 5 min in PBST, incubated with secondary antibodies in PBST for 1 h at room temperature (RT), washed 3 × 5 min in PBST, and mounted onto regular glass slides (Platinum Pro, Matsunami) with mounting medium with DAPI (Vectashield). After immunostaining, the mounted sections were observed and imaged under a confocal laser-scanning microscope (FV1200, Olympus). Primary antibodies against TH (Calbiochem, cat#657012, 1:1000 dilution), NeuN (Millipore, cat #MAB377, 1:1000 dilution), MAP2 (Sigma, cat # M4403, 1:200 dilution), GFAP (Dako, cat #Z0334, 1:1000 dilution), O4 (Chemicon international, cat #MAB345, 1:200 dilution), and Iba1 (Wako, cat #015-25191, 1:1000 dilution) were used. As secondary antibodies, donkey anti-mouse and donkey anti-rabbit labeled with Cy3 (Jackson ImmunoResearch) were used.

Histological analysis of SNCA protein in mouse brains

Brain samples were taken from PBS- and AmNa-ASO-treated Line 61 mice and wild type littermates. The hemispheres were divided at the midline. One hemisphere was fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for 2 hours at RT, cryoprotected in 15% sucrose in PBS overnight, embedded in OCT medium in cryomolds, and snap-frozen in dry ice-cooled liquid isopentane. Frozen samples were stored at −80 °C until sectioning. Frozen brain hemispheres were cryosectioned sagittally at 30 µm thickness on a Leica CM1850. Sections were transferred to 30% sucrose solution in PBS (Thermo Scientific) and stored at 4 °C. For immunostaining, sections were washed and incubated with primary and secondary antibodies as previously described in Furuya et al.52. In brief, the following primary antibodies were used: phosphorylated α-synuclein antibody, a mouse monoclonal antibody against α-synuclein phosphorylated at Ser129 (Wako, cat # 015-25191, 1:1000 dilution) and Syn211 (Invitrogen, AHB0261, 1:1000 dilution), and a mouse monoclonal antibody that specifically recognizes human α-synuclein. After immunostaining, mounted sections were observed and imaged under a fluorescence microscope (BZ-X700, Keyence).

ELISA for SNCA protein expression

Mouse brains were collected 30 days after injection. Mashed left brain hemispheres were homogenized in lysis buffer (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1% Triton X-100, 0.2 mM Sodium-orthovanadate, protease inhibitor cocktail (Calbiochem), and phosphatase inhibitor cocktail (Sigma)). Homogenates were incubated for 30 min on ice, followed by centrifugation at 15,000 g for 60 min at 4 °C. Supernatant was collected as the Triton X-100 soluble fraction. The Triton X-100-insoluble pellet was washed once in lysis buffer and then dissolved in lysis buffer containing 2% SDS. The resulting homogenate in 2% SDS was collected as the Triton X-100 insoluble fraction. hSNCA protein levels in the Triton X-100 soluble and insoluble fractions of all mice were determined using a commercially-available immunosorbent assay (Meso Scale Discovery, Cat#. K151TGD-4; Lot# K00E0147) according to the manufacturer's protocol. Plates were read on the Sector Imager 2400. For the assay, Triton X-100 soluble fractions were diluted 1:20000 and insoluble fractions 1:2000 in assay buffer. SNCA protein levels were evaluated in comparison to an adequate peptide standard as μg hSNCA and normalized to tissue weight (wet weight).

Statistical analysis

Dunnett's test was used to compare several treatments with a single control (e.g. Fig. 1a). One-way ANOVA, Tukey post hoc test was performed to compare the efficacy of control, ASO A19, and ASO L19 in HEK293 cells. Two-tailed unpaired t-tests were performed to compare the efficacy of control vs ASO A19 in mice. For all figures, the error bars represent the standard error of the mean (SEM). P-values below 0.05 were considered statistically significant.

Study approval

This research was approved by the institutional ethics committee. All of the procedures for animal experiments were approved by the institutional animal care and use committee of Osaka University.

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Acknowledgements

We acknowledge very useful comments from Gajanan Revankar and Kensuke Ikenaka. This study was supported by the Center for Medical Research and Education, Graduate School of Medicine, Osaka University. This work was supported by AMED under Grant Number JP17ek0109195. This work was supported by a part of the Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) from AMED and a part of the Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control) (15H01558) from MEXT.

Author information

Author notes

Takuya Uehara and Chi-Jing Choong contributed equally.

Affiliations

1. Graduate School of Medicine, Osaka University, Suita, Japan

Takuya Uehara, Chi-Jing Choong, Masayuki Nakamori, Hideki Hayakawa, Kumiko Nishiyama, Kousuke Baba, Hiroshi Tsuda & Hideki Mochizuki

2. National Institutes of Biomedical Innovation, Health and Nutrition, Suita, Japan

Yuuya Kasahara & Satoshi Obika

3. Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Tetsuya Nagata & Takanori Yokota

4. Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan

Yuuya Kasahara & Satoshi Obika

Contributions

M.N., Y.K., K.B., T.N., T.Y., S.O. and H.M. designed concept of this study. T.U., C.C., M.N., H.H., K.N., Y.K., H.T., S.O. and H.M. acquired data. T.U., C.C., M.N., H.H., K.N., Y.K., K.B., H.T., S.O. and H.M. analysed interpreted acquired data. T.U., C.C., M.N. and H.T. drafted the manuscript. M.N., H.H., Y.K., K.B., T.N., T.Y., H.T., S.O., K.I. and H.M. made critical revision.

Corresponding author

Correspondence to Hideki Mochizuki.

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Amido-bridged nucleic acid (AmNA)-modified antisense oligonucleotides targeting α-synuclein as a novel therapy for Parkinson's disease

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Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. A characteristic pathological feature of PD is cytoplasmic accumulation of α-synuclein (SNCA) protein. Multiplication of the SNCA gene in familial PD and pathological accumulation of SNCA protein during progression of sporadic PD suggest that increased SNCA protein levels increase the risk of PD.

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Detrimental Impacts of Astrocyte Depletion 

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Detrimental Impacts of Astrocyte Depletion 

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Highlights

*

When adult GFAP⁺ astrocytes are depleted in vivo, motor skills are severely impaired

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Neuronal loss occurs, whereas astroglial structural support still persists

*

Astroglial dysfunction disrupts CNS redox homeostasis, independent of microgliosis

*

Neutralization of ROS/RNS protects from neuronal injury

Summary

Although the importance of reactive astrocytes during CNS pathology is well established, the function of astroglia in adult CNS homeostasis is less well understood. With the use of conditional, astrocyte-restricted protein synthesis termination, we found that selective paralysis of GFAP⁺ astrocytes in vivo led to rapid neuronal cell loss and severe motor deficits. This occurred while structural astroglial support still persisted and in the absence of any major microvascular damage. Whereas loss of astrocyte function did lead to microglial activation, this had no impact on the neuronal loss and clinical decline. Neuronal injury was caused by oxidative stress resulting from the reduced redox scavenging capability of dysfunctional astrocytes and could be prevented by the in vivo treatment with scavengers of reactive oxygen and nitrogen species (ROS/RNS). Our results suggest that the subpopulation of GFAP⁺ astrocytes maintain neuronal health by controlling redox homeostasis in the adult CNS.

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Astrocyte Depletion Impairs Redox Homeostasis and Triggers Neuronal Loss in the Adult CNS

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When adult GFAP+ astrocytes are depleted in vivo, motor skills are severely impaired * Neuronal loss occurs, whereas astroglial structural support still persists * Astroglial dysfunction disrupts CNS redox homeostasis, independent of microgliosis * Neutralization of ROS/RNS protects from neuronal injury Although the importance of reactive astrocytes during CNS pathology is well established, the function of astroglia in adult CNS homeostasis is less well understood.