The role of activity in synaptic degeneration in a protein misfolding disease, prion disease

In chronic neurodegenerative diseases associated with aggregates of misfolded proteins (such as Alzheimer's, Parkinson's and prion disease), there is an early degeneration of presynaptic terminals prior to the loss of the neuronal somata. Identifying the mechanisms that govern synapse degeneration is of paramount importance, as cognitive decline is strongly correlated with loss of presynaptic terminals in these disorders. However, very little is known about the processes that link the presence of a misfolded protein to the degeneration of synapses. It has been suggested that the process follows a simple linear sequence in which terminals that become dysfunctional are targeted for death, but there is also evidence that high levels of activity can speed up degeneration. To dissect the role of activity in synapse degeneration, we infused the synaptic blocker botulinum neurotoxin A (BoNT/A) into the hippocampus of mice with prion disease and assessed synapse loss at the electron microscopy level. We found that injection of BoNT/A in naïve mice caused a significant enlargement of excitatory presynaptic terminals in the hippocampus, indicating transmission impairment. Long-lasting blockade of activity by BoNT/A caused only minimal synaptic pathology and no significant activation of microglia. In mice with prion disease infused with BoNT/A, rates of synaptic degeneration were indistinguishable from those observed in control diseased mice. We conclude that silencing synaptic activity neither prevents nor enhances the degree of synapse degeneration in prion disease. These results challenge the idea that dysfunction of synaptic terminals dictates their elimination during prion-induced neurodegeneration

Fibronectin impedes "myelin" sheet-directed flow in oligodendrocytes:A role for a beta 1 integrin-mediated PKC signaling pathway in vesicular trafficking

Differentiation of oligodendrocytes results in the formation of the myelin sheath, a dramatic morphological alteration that accompanies cell specialization. Here, we demonstrate that changes in the extracellular micro environment may regulate these morphological changes by altering intracellular vesicular trafficking of myelin sheet-directed proteins. The data reveal that fibronectin, in contrast to laminin-2, decreased membrane-directed transport of endogenous NCAM 140 and the model viral protein VSV G, both proteins normally residing in the myelin membrane. The underlying mechanism relies on an integrin-mediated activation of PKC, which causes stable phosphorylation of MARCKS. As a result, dynamic reorganization of the cortical actin cytoskeleton necessary for the targeting of vesicular trafficking to the myelin sheet is precluded, a prerequisite for morphological differentiation. These data are discussed in the context of the demyelinating disease multiple sclerosis, i.e., that leakage of fibronectin across the blood-brain barrier may impede myelination by interference with intracellular myelin sheet-directed membrane transport. (c) 2006 Elsevier Inc. All rights reserved

CRF and urocortin differentially modulate GluRdelta2 expression and distribution in parallel fiber-Purkinje cell synapses

Corticotropin-releasing factor (CRF) and urocortin (UCN) are closely related multifunctional regulators, governing, among other processes, Purkinje cell development. Here, we investigate the effects of CRF and UCN on Purkinje cells in organotypic slices. We show that both peptides upregulate delta2 ionotropic glutamate receptor gene expression, and increase the abundance of the receptor in the postsynaptic density. However, only UCN treatment results in increased delta2 protein level per Purkinje cell, implying the existence of posttranscriptional regulation of GluRdelta2 mRNA. CRF, in contrast, reduces the number of delta2-positive dendritic shafts per cell, implying that the increase of GluRdelta2 in remaining synapses may be mainly due to its retargeting. We further observed different patterns of GluRdelta2 distribution in the zone of postsynaptic density upon CRF and UCN treatment. CRF treatment results in a clustered distribution of GluRdelta2 along the postsynaptic density, whereas UCN treatment provides a linear distribution.status: publishe

Ultrastructural hallmarks of prion disease are not impacted by BoNT/A treatment.

<p>Electron micrographs of CA1 stratum radiatum of the hippocampus illustrating control, vehicle-exposed synapses (A, VEH) and neuropathological changes in prion+vehicle (prion + VEH; B) and prion+BoNT/A animals (C) at 12 weeks following delivery of the ME7 agent. High magnification pictures are shown in the insets. Note degenerating boutons with curved PSDs in both prion+vehicle and prion+BoNT/A samples. Scale bar  = 500 nm (250 nm for insets).</p

Neuropathological changes in a subset of BoNT/A-treated terminals at 4

<p> <b>weeks.</b> (A) Representative electron micrograph showing a degenerating, dark synaptic terminal with curved PSD. Scale bar  = 250 nm. (B) Percentage of degenerating synapses in vehicle (VEH)- and BoNT/A-treated animals. Dark terminals with a curved PSD (<120 deg) were scored as degenerating synapses. *, p<0.05. A total of 100 terminals were scored for each of three vehicle- and BoNT/A-injected animals at 4 weeks.</p

Ultrastructure of BoNT/A-treated hippocampal synapses.

<p>Representative electron micrographs of CA1 stratum radiatum in animals treated with vehicle (VEH) and at different times (2, 4 and 8 weeks) after BoNT/A infusion. There was no significant difference between the control groups at 2, 4 and 8 weeks after vehicle injection and the data from these groups has been merged. High magnification pictures are shown in the insets. Note enlarged presynaptic terminals following BoNT/A. Scale bar = 500 nm (250 nm for insets).</p

Lack of significant microglial activation 8 weeks after BoNT/A.

<p>(A, B) Representative immunostaining for the microglial marker Iba-1 in the CA1 stratum radiatum of mice treated with vehicle (A, VEH) and BoNT/A (B). Scale bar  = 100 µm. (C) Cell countings reveal no significant differences in microglial density between vehicle and BoNT/A-infused animals (t-test, p>0.05). (D, E) Immunostaining for cleaved SNAP-25 (red) demonstrates strong labeling in the CA1 stratum radiatum of BoNT/A-treated animals (E). No labeling is evident in vehicle mice (D). Green, Yoyo-1 nuclear counterstaining. Pyr, stratum pyramidale; s.r., stratum radiatum. Scale bar  = 100 µm.</p

BoNT/A has no significant impact on synaptic degeneration.

<p>(A) Percentages of healthy (open circles) and degenerating synapses (filled circles) in the CA1 stratum radiatum during prion disease progression. Degenerating terminals increase while normal boutons decrease in frequency from 10 to 16–18 weeks after delivery of the ME7 agent (one way ANOVA followed by Holm-Sidak test, p<0.05). Numbers of terminals analyzed are as follows: 10 weeks, n = 263; 12 weeks, n = 371; 16 weeks, n = 367; 18 weeks, n = 398. (B) Percentages of healthy (open bars) and degenerating synapses (filled bars) in prion+vehicle (prion+Veh) and prion+BoNT/A mice at 12 weeks into the disease. Percentages are identical in the two groups (t-test, p = 0.97). (C) Density of normal synapses (open bars) and degenerating boutons (filled bars) in prion+vehicle (prion+Veh) and prion+BoNT/A mice at 12 weeks into the disease. There are no statistically significant differences between the two groups (t-test, p>0.19). Number of terminals analyzed are as follows: prion+vehicle, n = 327; prion+BoNT/A, n = 723.</p