Aims

Abnormal cerebral α-synulcein accumulation plays an important role in synucleinopathy diseases such as Parkinson’ disease and dementia with Lewy body. There is a gap in non-invasive imaging of α-synulcein in animal models and in patients with synucleinopathy. Here, we developed a surface plamon resonance based high throughput screening pipeline and characterized probes for α-synulcein using recombinant fibrils and in animal models.

Methods

The binding properties of a panel of imaging probes to recombinant amyloid-beta (Aβ), 4-repeat K18 tau, and α-synuclein fibrils were assessed by using fluorescence assay using fluorometer and surface plasmon resonance assay. Immunofluorescence staining were performed using a panels of anti-Aβ, anti-phosphorylated tau, and anti-α-synuclein antibodies on brain slices from arcAβ and, APP/PS1 mice of amyloidosis, P301L mice of 4 repeat tau, A53T transgenic α-synuclein mice and non-transgenic littermates respectively.

Results

Among the 12 ligands we characterized, compound B showed a red-shift in fluorescence spectrum upon binding to α-synuclein fibril, where as no binding to Aβ and minor binding to K18 tau. Immunofluorescence staining in the brain slices from A53T mice demonstrated colocalization of compound B with ps129, and LB509, syn303 positive pathology.

Conclusions

We developed a high throughput binding property characterization method for ligands targeting Aβ, tau and α-synuclein to facilitate ligand development. Our results incidate that compound B detects α-synuclein specificaly and shows preference over 4 repeat tau and Aβ in vitro using recombinant fibrils and mouse brain slices, and is proming for in vivo investigation in animal models of α-synucleinopathy

Aims

Recent evidence suggests prion-like spreading of aggregated alpha-synuclein (a primary component of Lewy bodies, [aSyn]) mediates PD progression. We present a characterization of pathological aSyn spreading from a known locus via voxel-wise aSyn-induced changes in anatomy using magnetic resonance imaging (MRI).

Methods

11-week old wild-type (WT) and hemizygous M83 aSynA53T transgenic mice received an injection of either mouse [Ms-] or human [Hu-] aSyn preformed fibrils (PFF), or phosphate buffered saline (PBS) in the right striatum (n~10 mice/group/sex). T1-weighted MRI images (100 μm3 isotropic voxels; Bruker 7T) were acquired at -7, 30, 90, & 120 days post-injection (dpi). Brain atrophy was assessed using deformation-based morphometry. Linear mixed effects models were performed to examine injection by genotype differences in anatomy (voxel-wise).

Results

We observed significant changes in brain anatomy (steeper rates of change) in PFF mice, regardless of PFF species; with regions exhibiting volume increases (olfactory bulbs) and others decreases (corpus callosum, basal ganglia, motor cortices, thalamic nuclei) over time. M83-Ms-PFF mice exhibited steeper rates of change compared to WT and Hu-PFF mice; and concurrently exhibited earlier symptom onset, followed by M83-Hu-PFF, WT-Hu-PFF and lastly WT-Ms-PFF, coinciding with relatively stable rates of change for the WT-Ms-PFF mice in the aforementioned regions (Figure).

Conclusions

The PFF inoculation gives rise to widespread patterns of PFF-induced brain atrophy in regions that project to or receive input from the injection site. Presence of the mutation further modulates the degeneration. Symptomatology and consequently brain pathology appears to be more prominent and earlier onset in M83 Ms-PFF mice.

Aims

Our aim was to examine the possible neuroprotective effect of chronic rapamycin treatment in a rat model of cognitive dysfunction in Parkinson’s disease.

Methods

AAV-carrying-human-a-syn was stereotactically injected into substantia nigra and dentate gyri bilaterally and simultaneously in female SD rats. The a-syn injected and unlesioned animals were treated with either rapamycin i.p. (n=10, n=6) or vehicle (n=10, n=6) 3 days/week, starting from the first day of AAV injection until 8^th week. Further eight animals were used as naïve controls. Animals were tested for motor and cognitive functions between 16-18 weeks. Brain samples were semi-quantitatively analyzed for a-syn, synaptophysin, p62, LC3-II/I, beclin1 and pbeclin1 expression by immunoblotting (Ethics permission no:2014/51-08).

Results

Vehicle-treated a-syn group performed worse in Morris water maze compared to rapamycin-treated a-syn group, who performed almost equal to controls (p<0.05). But both a-syn groups spent less time with the novel object and showed worse motor performance compared to controls in novel object recognition and locomotor activity tests (p<0.05), respectively. Rapamycin treatment slightly decreased a-syn burden in both DG and SN. Hippocampal synaptophysin levels were lower in both a-syn groups, striatal synaptophysin level was lower only in vehicle-treated a-syn group compared to naïve controls. Autophagy activation markers beclin1, LC3-I -but not LC3-II- were increased with rapamycin treatment (p<0.05), whereas p62 or pbeclin1 levels didn’t change.

Conclusions

Rapamycin treatment improved the cognitive dysfunction due to a-syn overexpression by modulating autophagy at early stages of pathological process; loss of appetite and generalized weakness due to rapamycin may be responsible from unexpected motor worsening.

Aims

Lowering alpha-synuclein or eliminating toxic alpha-synuclein species in cells could be a therapeutic approach to treat Parkinson’s disease or related alpha-synucleinopathies. This study translated our in vitro CRISPRi-mediated alpha-synuclein downregulation to in vivo studies in a P1-derived artificial chromosome SNCA A53T transgenic mouse model. The study aimed at testing the route of administration for Cas9 transgene expression and alpha-synuclein knockdown.

Methods

We tested a CRISPRi/guide RNA expression cassette in an adeno-associated virus AAV9 system with a neuron-specific MECP2 promoter, including sham controls and controls expressing nonsense guide RNA. We tested two routes of administration that were cisterna magna injections and stereotactic injections into the substantia nigra. Animals were between 4-5 months, and we assessed the expression of the transgene 1-month post-surgery. Titer was adjusted in both administration routes to 1.5 X 10¹⁰ GC/ml. We analyzed the tissue with multiplex immunofluorescense imaging CODEX and in-situ hybridization RNAScope.

Results

Cas9 is co-expressed with in NEUN-positive neurons 1 month after surgery. Via intrathecal injection, Cas9 is detected in the cerebellum, main olfactory bulb, striatum, and in the mesencephalon.

We optimized the quantification of alpha-synuclein and Cas9 mRNA expression with RNAScope technology. With CODEX multiplexing, we can also assess inflammatory response of the transgene.

Conclusions

Alpha-synuclein has also become a leading target for the development of new therapies aimed at disease modification in Parkinson’s. With these studies, we can test both alpha-synuclein lowering strategies and CRISPR/Cas9 as a therapeutic strategy in one model.

Abstract Body

Aims: Exposure to low doses of carbon monoxide (CO) may underlie the reduced risk of Parkinson’s disease (PD) among smokers. Here, we set out to evaluate the neuroprotective potential of low-dose CO treatment in PD models.

Methods: In the AAV-alpha-synuclein (aSyn) model, rats underwent right nigral injection of AAV1/2- asynA53T and left injection of empty AAV, and were treated with oral CO drug product (HBI-002 10ml/kg, daily by gavage) or vehicle. HBI-002 is under development by Hillhurst Pharmaceuticals. In the short-term MPTP model (40mg/kg, i.p.), mice were treated with inhaled CO (iCO) (250ppm) or air. HPLC measurement of striatal dopamine, immunohistochemistry for nigral aSyn and tyrosine hydroxylase, stereological cell counting, and biochemical analyses were conducted blinded to treatment condition.

Results: Each HBI-002 treatment increased carboxy-hemoglobin to 6%. Administration of HBI-002 in the aSyn model reduced ipsilateral loss of both striatal dopamine and TH-positive neurons in the substantia nigra pars compacta compared to rats treated with HBI-002 vehicle. HBI-002 reduced aSyn aggregates and S129 phosphorylation. MPTP-exposed mice treated with low dose iCO had higher dopamine levels and more TH+ neurons than those treated with air. In saline-treated mice, iCO had no effect on striatal dopamine levels or TH+ cell counts. HBI-002 upregulated heme oxygenase-1 and HIF-1α.

Conclusions: These results demonstrating reduced dopamine cell death and aSyn pathology advance low dose CO as a potential neuroprotective strategy for PD.