Soluble amyloid beta levels are elevated in the white matter of Alzheimer’s patients, independent of cortical plaque severity

Alzheimer’s disease (AD) is the most common neurodegenerative disease and the leading cause of dementia. In addition to grey matter pathology, white matter changes are now recognized as an important pathological feature in the emergence of the disease. Despite growing recognition of the importance of white matter abnormalities in the pathogenesis of AD, the causes of white matter degeneration are still unknown. While multiple studies propose Wallerian-like degeneration as the source of white matter change, others suggest that primary white matter pathology may be due, at least in part, to other mechanisms, including local effects of toxic Aβ peptides. In the current study, we investigated levels of soluble amyloid-beta (Aβ) in white matter of AD patients (n=12) compared with controls (n=10). Fresh frozen white matter samples were obtained from anterior (Brodmann area 9) and posterior (Brodmann area 1, 2 and 3) areas of post-mortem AD and control brains. ELISA was used to examine levels of soluble Aβ -42 and Aβ -40. Total cortical neuritic plaque severity rating was derived from individual ratings in the following areas of cortex: mid-frontal, superior temporal, pre-central, inferior parietal, hippocampus (CA1), subiculum, entorhinal cortex, transentorhinal cortex, inferior temporal, amygdala and basal forebrain. Compared with controls, AD samples had higher white matter levels of both soluble Aβ -42 and Aβ -40. While no regional white matter differences were found in Aβ -40, Aβ -42 levels were higher in anterior regions than in posterior regions across both groups. After statistically controlling for total cortical neuritic plaque severity, differences in both soluble Aβ -42 and Aβ -40 between the groups remained, suggesting that white matter Aβ peptides accumulate independent of overall grey matter fibrillar amyloid pathology and are not simply a reflection of overall amyloid burden. These results shed light on one potential mechanism through which white matter degeneration may occur in AD. Given that white matter degeneration may be an early marker of disease, preceding grey matter atrophy, understanding the mechanisms and risk factors that may lead to white matter loss could help to identify those at high risk and to intervene earlier in the pathogenic process

Dynamin 1 is required for memory formation.

Dynamin 1-3 isoforms are known to be involved in endocytotic processes occurring during synaptic transmission. No data has directly linked dynamins yet with normal animal behavior. Here we show that dynamin pharmacologic inhibition markedly impairs hippocampal-dependent associative memory. Memory loss was associated with changes in synaptic function occurring during repetitive stimulation that is thought to be linked with memory induction. Synaptic fatigue was accentuated by dynamin inhibition. Moreover, dynamin inhibition markedly reduced long-term potentiation, post-tetanic potentiation, and neurotransmitter released during repetitive stimulation. Most importantly, the effect of dynamin inhibition onto memory and synaptic plasticity was due to a specific involvement of the dynamin 1 isoform, as demonstrated through a genetic approach with siRNA against this isoform to temporally block it. Taken together, these findings identify dynamin 1 as a key protein for modulation of memory and release evoked by repetitive activity

Dynamin inhibition by dynasore affects LTP, a type of synaptic plasticity due to sustained activity in hippocampus.

<p><b>A</b>, Dynasore (80 μM, 20 minute perfusion, open triangles) decreases LTP induced by theta-burst stimulation in CA₃–CA₁ synapses compared to vehicle-treated slices (black circles)(F_1,10 = 9.081, <i>p</i> = 0.013). The horizontal bar indicates the period of perfusion with dynasore before tetanic stimulation. <b>B</b>, Post-tetanus dynamin inhibition by dynasore (80 μm, 20 minute perfusion <i>after</i> the tetanus delivery, open triangle) induced by theta-burst stimulation in CA₃–CA₁ synapses compared to vehicle-treated slices (black circles; F_1,7 = 0.209, <i>p</i> = 0.662). The horizontal bar indicates the period of perfusion with dynasore <i>after</i> tetanic stimulation. <b>C</b>, Basal synaptic transmission is unmodified by dynamin inhibition with dynasore. Averaged evoked field potential slopes as a function of stimulation intensity measured in volts (V) at CA₃–CA₁ synapses in slices do not show significant differences between vehicle-treated (black circles) and dynasore (80 μM, open triangles) treated slices (F_1,11 = 40.081, <i>p</i> = 0.7013). <b>D</b>, Dynamin inhibition by dynasore (open triangles; 80 μm, 20 minute perfusion before the tetanus) does not produce changes in solely post-synaptic LTP induced by three tetani at 50 Hz for 1 second, each tetanus separated by 20 seconds, at the CA₃–CA₁ synapse compared to vehicle-treated slices (black circles; F1,8 = 1.538, p = 0.250). The horizontal bar indicates the period of perfusion with dynasore before tetanic stimulation. Error bars indicate SEM.</p

Dynamin inhibition by dynasore increases synaptic fatigue following sustained activity in hippocampus.

<p><b>A</b>, Dynasore treatment (80 μM, open triangles) increases SF in slices that were previously treated with vehicle (black circles) (F_1,12 = 6.395, <i>p</i> = 0.026). The increase is already present at the 10^th pulse during the stimulation (Mann-Whitney U_{69, 36} = 8.00, <i>p</i> = 0.0379). The effect was reversed by washout with vehicle (open squares). SF was induced by high frequency stimulation in the presence of D-APV (100 μM). <b>B</b>, SF was induced by high frequency stimulation (100 Hz, 1 second) in slices containing both D-APV (100 μM) and cyclothiazide (100 μM). Dynasore (80 μM, open triangles) further increases SF compared to fatigue of the same slices in the presence of vehicle (black circles) (F_1,12 = 6.395, <i>p</i> = 0.026). SF increases in dynasore-treated slices already at the 10^th pulse during the tetanus (Mann-Whitney U_{35, 10} = 0.0001, <i>p</i> = 0.0159). The effect is reversed by washout with vehicle (open squares), re-establishing SF to the values obtained prior to dynasore perfusion. <b>C</b>, SF is induced by high frequency stimulation (100 Hz, 1 second) in vehicle-treated slices (black circles) containing both D-APV (100 μM), the GABA_A receptor blocker picrotoxin (30 μM), the GABA_B receptor blocker SCH 50911 (100 μM). Dynasore (80 μM, open triangles) further increases SF compared to fatigue of the same slices in the presence of vehicle (black circles) (F_1,12 = 6.395, <i>p</i> = 0.026). SF increases in dynasore-treated slices already at the 10^th pulse during the tetanus (Mann-Whitney U_{159, 94} = 28.00, <i>p</i> = 0.0356). The effect is reversed by washout with vehicle (open squares), re-establishing SF to the values obtained prior to dynasore perfusion. Data shows dynasore-induced increase in SF is not associated to AMPA receptor desensitization or changes in GABA_A/B responsiveness. Error bars indicate SEM.</p

Dynamin inhibition affects presynaptic mechanisms underlying synaptic plasticity evoked by sustained activity.

<p><b>A</b>, Tetanic efficiency, expressed as percent in evoked potential area across a train of 10 bursts at 5 Hz, each consisting of 4 pulses at 100 Hz, is reduced by dynasore (80 μM, open bars) with respect to percent response area in vehicle-treated slices (black bars) (F_1,9 = 12.071, <i>p</i> = 0.007). The reduction reaches statistical significance at the third burst (Mann-Whitney U_{51, 15} = 0.00001, <i>p</i> = 0.0043); <b>B</b>, Dynasore (80 μM, open bars) already produces a partial reduction of the area within the first group of 4 pulses at 100 Hz compared to vehicle-treated slices (black bars); <b>C</b>, Raw <i>f</i>EPSP signals recorded during tetanic stimulation in vehicle-treated and dynasore (80 μM, 20 minutes before tetanus)-treated hippocampal slices. Calibration: 0.5 mV, 2 ms;. <b>D</b>, Perfusion of hippocampal slices with dynasore (80 μM) in the presence of D-APV (100 μM) for 20 minutes prior to theta-burst stimulation diminishes PTP to about 50% of the values obtained with vehicle perfusion (F_1,12 = 6.924, <i>p</i> = 0.022). The effect was reversed by washout with vehicle. Error bars indicate SEM.</p

Dynamin inhibition with dynasore does not affect paired-pulse facilitation, a type of synaptic plasticity that is not linked with sustained activity.

<p>Paired-pulse-induced change is calculated as the ratio of the slope of the second evoked field potential to the first one at different interpulse intervals (1–1000 ms). Paired-pulse ratio is not modified in dynasore (80 μM, open triangles) treated slices with respect to vehicle-treated slices (black circles)(F1,12 = 0.339, p = 0.914). No effect is observed by subsequent washout with ACSF (open squares). Error bars indicate SEM.</p

Selective dynamin 1 inhibition through siRNA impairs both synaptic plasticity and associative memory.

<p><b>A</b>, siRNA specific for murine dynamin 1 reduces protein expression. An example of western blot showing that Penetratin 1- conjugated dynamin 1 siRNA reduces protein expression. Cells are lysed 48 hours after the treatment with siRNA. Dynamin 1 is detected using a rabbit polyclonal anti-dynamin1 antibody. Penetratin 1- conjugated Control siRNA, that does not affect dynamin 1 expression, does not change protein levels. n = 3 for each group. <b>B</b>, Penetratin 1- conjugated dynamin 1 siRNA (open bars) (80 nM in a final volume of 1.5 μl over 1 minute, bilateral injections twice a day for 3 days) impairs contextual fear memory compared to control siRNA infused mice (grey bars) (Mann-Whitney U_{150, 126} = 21.00, <i>p</i> = 0.0089). Moreover, control siRNA infused mice show similar amount of freezing as vehicle-infused animals (black bars). <b>C</b>, Penetratin 1- conjugated dynamin 1 siRNA (open bars) does not modify cued fear memory compared to Penetratin 1- conjugated Control siRNA (grey bars) (Mann-Whitney U_{99, 90} = 44.50, <i>p</i> = 1.00) in mice previously tested for contextual fear conditioning at 24 hours after the shock. <b>D</b>, Bilateral infusions of Penetratin 1- conjugated dynamin 1 siRNA (open triangles) (80 nM in a final volume of 1.5 μl over 1 minute, repeated 2 times a day for three days) into dorsal hippocampi decrease LTP compared to Penetratin 1- conjugated Control siRNA treatment (grey squares) (F_1,9 = 5.578, <i>p</i> = 0.001). As an internal control, slices from vehicle-infused animals (black circles) show similar amounts of potentiation as those from Penetratin 1- conjugated Control siRNA treated animals. Error bars indicate SEM.</p

Dynamin inhibition by dynasore impairs associative memory.

<p><b>A</b>, Schematic representation of the hippocampi bilaterally implanted with cannulas. <b>B</b>, Bilateral injections of dynasore (80 μM in a final volume of 1.5 μl over 1 minute) into dorsal hippocampi, 20 minutes before training, dramatically impairs contextual fear memory (open bars) compared to vehicle treated mice (black bars) (Mann-Whitney U_{137, 73} = 18.00, <i>p</i> = 0.00147). <b>C</b>, Mice do not show changes in cued fear conditioning following dynasore infusions (open bars) compared to vehicle-treated animals (black bars) (Mann-Whitney U_{121, 89} = 34.00, <i>p</i> = 0.2412). Each bar represents the average percent of time spent in freezing posture. Error bars indicate SEM. <b>D</b>, Sensory threshold was not affected regardless of treatment (n = 10).</p