Edited by Phyllis I. Hanson Extracellular vesicles (EVs) are secreted by myriad cells in culture and also by unicellular organisms, and their identification in mammalian fluids suggests that EV release also occurs at the organism level. However, although it is clearly important to better understand EVs' roles in organismal biology, EVs in solid tissues have received little attention. Here, we modified a protocol for EV isolation from primary neural cell culture to collect EVs from frozen whole murine and human neural tissues by serial centrifugation and purification on a sucrose gradient. Quantitative proteomics comparing brain-derived EVs from nontransgenic (NTg) and a transgenic amyotrophic lateral sclerosis (ALS) mouse model, superoxide dismutase 1 (SOD1) G93A , revealed that these EVs contain canonical exosomal markers and are enriched in synaptic and RNA-binding proteins. The compiled brain EV proteome contained numerous proteins implicated in ALS, and EVs from SOD1 G93A mice were significantly depleted in myelin-oligodendrocyte glycoprotein compared with those from NTg animals. We observed that brain-and spinal cord-derived EVs, from NTg and SOD1 G93A mice, are positive for the astrocyte marker GLAST and the synaptic marker SNAP25, whereas CD11b, a microglial marker, was largely absent. EVs from brains and spinal cords of the SOD1 G93A ALS mouse model, as well as from human SOD1 familial ALS patient spinal cord, contained abundant misfolded and nonnative disulfide-cross-linked aggregated SOD1. Our results indicate that CNS-derived EVs from an ALS animal model contain pathogenic disease-causing proteins and suggest that brain astrocytes and neurons, but not microglia, are the main EV source. This work was supported by a Bernice Ramsay ALS Canada grant, along with funding from the Paul Heller Memorial Fund (to J. M. S.). N. R. C. is the Chief Scientific Officer of ProMIS Neurosciences, which has licensed the 3H1 misfolded SOD1-specific antibody technology. This article contains Figs. S1 and S2 and Tables S1-S9.
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neuromuscular degenerative disorder with a poorly defined etiology. ALS patients experience motor weakness, which starts focally and spreads throughout the nervous system, culminating in paralysis and death within a few years of diagnosis. While the vast majority of clinical ALS is sporadic with no known cause, mutations in human copper-zinc superoxide dismutase 1 (SOD1) cause about 20 % of inherited cases of ALS. ALS with SOD1 mutations is caused by a toxic gain of function associated with the propensity of mutant SOD1 to misfold, presenting a non-native structure. The mechanisms responsible for the progressive spreading of ALS pathology have been the focus of intense study. We have shown that misfolded SOD1 protein can seed misfolding and aggregation of endogenous wild-type SOD1 similar to amyloid-β and prion protein seeding. Our recent observations demonstrate a transfer of the misfolded SOD1 species from cell to cell, modeling the intercellular transmission of disease through the neuroaxis. We have shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell, either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or in association with vesicles which are released into the extracellular environment. Furthermore, once misfolding of wild-type SOD1 has been initiated in a human cell culture, it can induce misfolding in naïve cell cultures over multiple passages of media transfer long after the initial misfolding template is degraded. Herein we review the data on mechanisms of intercellular transmission of misfolded SOD1.
SummaryThe secreted growth factor progranulin (PGRN) has been shown to be important for regulating neuronal survival and outgrowth, as well as synapse formation and function. Mutations in the PGRN gene that result in PGRN haploinsufficiency have been identified as a major cause of frontotemporal dementia (FTD). Here we demonstrate that PGRN is colocalized with dense-core vesicle markers and is cotransported with brain-derived neurotrophic factor (BDNF) within axons and dendrites of cultured hippocampal neurons in both anterograde and retrograde directions. We also show that PGRN is secreted in an activity-dependent manner from synaptic and extrasynaptic sites, and that the temporal profiles of secretion are distinct in axons and dendrites. Neuronal activity is also shown to increase the recruitment of PGRN to synapses and to enhance the density of PGRN clusters along axons. Finally, treatment of neurons with recombinant PGRN is shown to increase synapse density, while decreasing the size of the presynaptic compartment and specifically the number of synaptic vesicles per synapse. Together, this indicates that activity-dependent secretion of PGRN can regulate synapse number and structure.
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