Novel systemic delivery of a peptide-conjugated antisense oligonucleotide to reduce α-synuclein in a mouse model of Alzheimer's disease.

Neurodegenerative disorders of aging are characterized by the progressive accumulation of proteins such as α-synuclein (α-syn) and amyloid beta (Aß). Misfolded and aggregated α-syn has been implicated in neurological disorders such as Parkinson's disease, and Dementia with Lewy Bodies, but less so in Alzheimer's Disease (AD), despite the fact that accumulation of α-syn has been confirmed in over 50% of postmortem brains neuropathologically diagnosed with AD. To date, no therapeutic strategy has effectively or consistently downregulated α-syn in AD. Here we tested the hypothesis that by using a systemically-delivered peptide (ApoB11) bound to a modified antisense oligonucleotide against α-syn (ASO-α-syn), we can downregulate α-syn expression in an AD mouse model and improve behavioral and neuropathologic phenotypes. Our results demonstrate that monthly systemic treatment with of ApoB11:ASO α-syn beginning at 6 months of age reduces expression of α-synuclein in the brains of 9-month-old AD mice. Downregulation of α-syn led to reduction in Aß plaque burden, prevented neuronal loss and astrogliosis. Furthermore, we found that AD mice treated with ApoB11:ASO α-syn had greatly improved hippocampal and spatial memory function in comparison to their control counterparts. Collectively, our data supports the reduction of α-syn through use of systemically-delivered ApoB11:ASO α-syn as a promising future disease-modifying therapeutic for AD.

Hippocampal Reduction of α-Synuclein via RNA Interference Improves Neuropathology in Alzheimer's Disease Mice.

BACKGROUND: Alzheimer's disease (AD) cases are often characterized by the pathological accumulation of α-synuclein (α-syn) in addition to amyloid-ß (Aß) and tau hallmarks. The role of α-syn has been extensively studied in synucleinopathy disorders, but less so in AD. Recent studies have shown that α-syn may also play a role in AD and its downregulation may be protective against the toxic effects of Aß accumulation. OBJECTIVE: We hypothesized that selectively knocking down α-syn via RNA interference improves the neuropathological and biochemical findings in AD mice. METHODS: Here we used amyloid precursor protein transgenic (APP-Tg) mice to model AD and explore pathologic and behavioral phenotypes with knockdown of α-syn using RNA interference. We selectively reduced α-syn levels by stereotaxic bilateral injection of either LV-shRNA α-syn or LV-shRNA-luc (control) into the hippocampus of AD mice. RESULTS: We found that downregulation of α-syn results in significant reduction in the number of Aß plaques. In addition, mice treated with LV-shRNA α-syn had amelioration of abnormal microglial activation (Iba1) and astrocytosis (GFAP) phenotypes in AD mice. CONCLUSION: Our data suggests a novel link between Aß and α-syn pathology as well as a new therapeutic angle for targeting AD.

Overexpression of alpha synuclein disrupts APP and Endolysosomal axonal trafficking in a mouse model of synucleinopathy.

Mutations or triplication of the alpha synuclein (ASYN) gene contribute to synucleinopathies including Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Recent evidence suggests that ASYN also plays an important role in amyloid-induced neurotoxicity, although the mechanism(s) remains unknown. One hypothesis is that accumulation of ASYN alters endolysosomal pathways to impact axonal trafficking and processing of the amyloid precursor protein (APP). To define an axonal function for ASYN, we used a transgenic mouse model of synucleinopathy that expresses a GFP-human ASYN (GFP-hASYN) transgene and an ASYN knockout (ASYN-/-) mouse model. Our results demonstrate that expression of GFP-hASYN in primary neurons derived from a transgenic mouse impaired axonal trafficking and processing of APP. In addition, axonal transport of BACE1, Rab5, Rab7, lysosomes and mitochondria were also reduced in these neurons. Interestingly, axonal transport of these organelles was also affected in ASYN-/- neurons, suggesting that ASYN plays an important role in maintaining normal axonal transport function. Therefore, selective impairment of trafficking and processing of APP by ASYN may act as a potential mechanism to induce pathological features of Alzheimer's disease (AD) in PD patients.

Endosomal sorting drives the formation of axonal prion protein endoggresomes.

The pathogenic aggregation of misfolded prion protein (PrP) in axons underlies prion disease pathologies. The molecular mechanisms driving axonal misfolded PrP aggregate formation leading to neurotoxicity are unknown. We found that the small endolysosomal guanosine triphosphatase (GTPase) Arl8b recruits kinesin-1 and Vps41 (HOPS) onto endosomes carrying misfolded mutant PrP to promote their axonal entry and homotypic fusion toward aggregation inside enlarged endomembranes that we call endoggresomes. This axonal rapid endosomal sorting and transport-dependent aggregation (ARESTA) mechanism forms pathologic PrP endoggresomes that impair calcium dynamics and reduce neuronal viability. Inhibiting ARESTA diminishes endoggresome formation, rescues calcium influx, and prevents neuronal death. Our results identify ARESTA as a key pathway for the regulation of endoggresome formation and a new actionable antiaggregation target to ameliorate neuronal dysfunction in the prionopathies.

Selectivity of Lewy body protein interactions along the aggregation pathway of α-synuclein.

The aggregation of alpha-synuclein (α-SYN) follows a cascade of oligomeric, prefibrillar and fibrillar forms, culminating in the formation of Lewy Bodies (LB), the pathological hallmarks of Parkinson's Disease. Although LB contain over 70 proteins, the potential for interactions along the aggregation pathway of α-SYN is unknown. Here we propose a map of interactions of 65 proteins against different species of α-SYN. We measured binding to monomeric α-SYN using AlphaScreen, a sensitive nano-bead luminescence assay for detection of protein interactions. To access oligomeric species, we used the pathological mutants of α-SYN (A30P, G51D and A53T) which form oligomers with distinct properties. Finally, we generated amyloid fibrils from recombinant α-SYN. Binding to oligomers and fibrils was measured by two-color coincidence detection (TCCD) on a single molecule spectroscopy setup. Overall, we demonstrate that LB components are recruited to specific steps in the aggregation of α-SYN, uncovering future targets to modulate aggregation in synucleinopathies.