Explore the Role of Abeta in Axonal Trafficking Deficits Induced by Alpha Synuclein in Parkinson Disease Mouse Model

Alpha synuclein (ASYN) is a neuronal protein that is observed in significant amounts in the brain and is encoded for by the SNCA gene, it functions as a regulator for the trafficking of synaptic vesicles. It has been noted that the buildup of alpha synuclein has been found in the form of Lewy bodies in studies involving patients with Parkinson’s diseases (PD). Gathering an understanding for the manner in which alpha synuclein affects the synaptic structure and the movement of axonal trafficking will help further our understanding towards the formation of Lewy bodies. Experimenting with the way in which ASYN affected the intervention of Abeta was important, to see the toxicity of Abeta in axonal trafficking. The PD and SynKO mouse models treated with Abeta both showed an effect on the anterograde moving speed of both the PD and SynKO neurons. Synaptic formation was examined, and it was found that ASYN had a large negative influence on the synapse formation in PD neurons. This was due to the significantly reduced colocalization that was found in the treated neurons. It was confirmed that ASYN caused neuronal atrophy through the over expression of GFP-ASYNWT wild type or the GFP-ASYNA53T. Comprehending ASYN effect on the axonal trafficking and the synaptic structure of PD neurons can help understand the mechanism that may be present which possibly stimulates Alzheimer’s Disease in PD patients.

Effect of G4C2-Repeat Expansions on the Motion of Lysosomes Inside Neurites.

The G4C2 hexanucleotide repeat expansion in the c9orf72 locus is one among a plethora of mutations associated with amyotrophic lateral sclerosis. It accounts for the majority of disease cases. The exact processes underlying the pathology of this mutation remain elusive, yet recent evidence suggests a mechanism that disrupts axonal trafficking. Here, we used a neuronal cell line with and without the G4C2 repeats, and implemented time-resolved local mean squared displacement analysis to characterize the motion of lysosomes inside neurites. Neurites were either aligned along chemically patterned lines, or oriented randomly on the substrate. We confirmed that in the presence of the G4C2 repeats, lysosome motion was affected. Lysosomes had a smaller reach exhibited lower velocity, especially inside aligned neurites. At the same time they became more active with increasing length of the G4C2 repeats when the neurites were randomly oriented. The duration of diffusive and super-diffusive lysosome transport remained unaffected for both neurite geometries and for all lengths of the repeats, but the displacement and velocity was decreased on varying the repeat number and neurite geometry. Lastly, the ratio of anterograde/retrograde/neutral trajectories was affected disparately for the two neurite geometries. Our observations support the hypothesis that impaired axonal trafficking emerges in the presence of the G4C2 hexanucleotide repeat expansion.

C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell–derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult Drosophila neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to potential therapeutic strategies.

SCAMP5 plays a critical role in axonal trafficking and synaptic localization of NHE6 to adjust quantal size at glutamatergic synapses

Glutamate uptake into synaptic vesicles (SVs) depends on cation/H+ exchange activity, which converts the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane at the presynaptic terminals. Thus, the proper recruitment of cation/H+ exchanger to SVs is important in determining glutamate quantal size, yet little is known about its localization mechanism. Here, we found that secretory carrier membrane protein 5 (SCAMP5) interacted with the cation/H+ exchanger NHE6, and this interaction regulated NHE6 recruitment to glutamatergic presynaptic terminals. Protein–protein interaction analysis with truncated constructs revealed that the 2/3 loop domain of SCAMP5 is directly associated with the C-terminal region of NHE6. The use of optical imaging and electrophysiological recording showed that small hairpin RNA–mediated knockdown (KD) of SCAMP5 or perturbation of SCAMP5/NHE6 interaction markedly inhibited axonal trafficking and the presynaptic localization of NHE6, leading to hyperacidification of SVs and a reduction in the quantal size of glutamate release. Knockout of NHE6 occluded the effect of SCAMP5 KD without causing additional defects. Together, our results reveal that as a key regulator of axonal trafficking and synaptic localization of NHE6, SCAMP5 could adjust presynaptic strength by regulating quantal size at glutamatergic synapses. Since both proteins are autism candidate genes, the reduced quantal size by interrupting their interaction may underscore synaptic dysfunction observed in autism.

Gga3 deletion and a GGA3 rare variant associated with late onset Alzheimer’s disease trigger BACE1 accumulation in axonal swellings

Axonal dystrophy, indicative of perturbed axonal transport, occurs early during Alzheimer’s disease (AD) pathogenesis. Little is known about the mechanisms underlying this initial sign of the pathology. This study proves that Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) loss of function, due to Gga3 genetic deletion or a GGA3 rare variant that cosegregates with late-onset AD, disrupts the axonal trafficking of the β-site APP-cleaving enzyme 1 (BACE1) resulting in its accumulation in axonal swellings in cultured neurons and in vivo. We show that BACE pharmacological inhibition ameliorates BACE1 axonal trafficking and diminishes axonal dystrophies in Gga3 null neurons in vitro and in vivo. These data indicate that axonal accumulation of BACE1 engendered by GGA3 loss of function results in local toxicity leading to axonopathy. Gga3 deletion exacerbates axonal dystrophies in a mouse model of AD before β-amyloid (Aβ) deposition. Our study strongly supports a role for GGA3 in AD pathogenesis, where GGA3 loss of function triggers BACE1 axonal accumulation independently of extracellular Aβ, and initiates a cascade of events leading to the axonal damage distinctive of the early stage of AD.

Axon Injury-Induced Autophagy Activation Is Impaired in a C. elegans Model of Tauopathy

Autophagy is a conserved pathway that plays a key role in cell homeostasis in normal settings, as well as abnormal and stress conditions. Autophagy dysfunction is found in various neurodegenerative diseases, although it remains unclear whether autophagy impairment is a contributor or consequence of neurodegeneration. Axonal injury is an acute neuronal stress that triggers autophagic responses in an age-dependent manner. In this study, we investigate the injury-triggered autophagy response in a C. elegans model of tauopathy. We found that transgenic expression of pro-aggregant Tau, but not the anti-aggregant Tau, abolished axon injury-induced autophagy activation, resulting in a reduced axon regeneration capacity. Furthermore, axonal trafficking of autophagic vesicles were significantly reduced in the animals expressing pro-aggregant F3ΔK280 Tau, indicating that Tau aggregation impairs autophagy regulation. Importantly, the reduced number of total or trafficking autophagic vesicles in the tauopathy model was not restored by the autophagy activator rapamycin. Loss of PTL-1, the sole Tau homologue in C. elegans, also led to impaired injury-induced autophagy activation, but with an increased basal level of autophagic vesicles. Therefore, we have demonstrated that Tau aggregation as well as Tau depletion both lead to disruption of injury-induced autophagy responses, suggesting that aberrant protein aggregation or microtubule dysfunction can modulate autophagy regulation in neurons after injury.

High content live profiling reveals concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis

Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS), a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOF) through DPRs versus loss of C9ORF72 functions (LOF). Through multiparametric HC live profiling in spinal MNs from induced pluripotent stem cells (iPSCs) and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA strand breaks (DSBs), DPRs and apoptosis. We show that both GOF and LOF were necessary to yield the overall C9ORF72 pathology. Finally, C9ORF72 LOF was sufficient – albeit to a smaller extent – to induce proximal axonal trafficking deficits and increased DSBs.Single sentence summaryPathogenesis in C9ORF72 ALS shows a distinct spatiotemporal axonal organelle trafficking impairment caused by gain and loss of function mechanisms.