Singh A et al. (2020), Functional Integrity of Synapses in the Central...

XB-ART-57525

J Alzheimers Dis 2020 Jan 01;784:1661-1678. doi: 10.3233/JAD-200716.

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Functional Integrity of Synapses in the Central Nervous System of Cognitively Intact Individuals with High Alzheimer's Disease Neuropathology Is Associated with Absence of Synaptic Tau Oligomers.

Singh A , Allen D , Fracassi A , Tumurbaatar B , Natarajan C , Scaduto P , Woltjer R , Kayed R , Limon A , Krishnan B , Taglialatela G .

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BACKGROUND: Certain individuals, here referred to as Non-Demented with Alzheimer Neuropathology (NDAN), do not show overt neurodegeneration (N-) and remain cognitively intact despite the presence of plaques (A+) and tangles (T+) that would normally be consistent with fully symptomatic Alzheimer's disease (AD). OBJECTIVE: The existence of NDAN (A + T+N-) subjects suggests that the human brain utilizes intrinsic mechanisms that can naturally evade cognitive decline normally associated with the symptomatic stages of AD (A + T+N+). Deciphering the underlying mechanisms would prove relevant to develop complementing therapeutics to prevent progression of AD-related cognitive decline. METHODS: Previously, we have reported that NDAN present with preserved neurogenesis and synaptic integrity paralleled by absence of amyloid oligomers at synapses. Using postmortem brain samples from age-matched control subjects, demented AD patients and NDAN individuals, we performed immunofluorescence, western blots, micro transplantation of synaptic membranes in Xenopus oocytes followed by twin electrode voltage clamp electrophysiology and fluorescence assisted single synaptosome-long term potentiation studies. RESULTS: We report decreased tau oligomers at synapses in the brains of NDAN subjects. Furthermore, using novel approaches we report, for the first time, that such absence of tau oligomers at synapses is associated with synaptic functional integrity in NDAN subjects as compared to demented AD patients. CONCLUSION: Overall, these results give further credence to tau oligomers as primary actors of synaptic destruction underscoring cognitive demise in AD and support their targeting as a viable therapeutic strategy for AD and related tauopathies.

???displayArticle.pubmedLink??? 33185603
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???displayArticle.link??? J Alzheimers Dis
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Genes referenced: ctrl dlg4 psd sia2

???displayArticle.disOnts??? Alzheimer's disease
???displayArticle.omims??? ALZHEIMER DISEASE, FAMILIAL, 1; AD1

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	Fig. 1. Reduced levels of toxic tau oligomers in hippocampus and frontal cortex of NDAN subjects. A) Representative immunofluorescence images of Ctrl, AD, and NDAN frontal cortex showing Tau5 (green), T22 (red), and merge including DAPI (blue), bar scale: 100μm. B) Histogram summarizing quantitation of tau oligomer (T22) immunostaining in Ctrl, AD, and NDAN. T22 expression was significantly higher in the AD group (**p < 0.0001) compared to either Ctrl or NDAN and interestingly, T22 expression was also increased in the NDAN group (p < 0.0001) compared to Ctrl (n = 4 subjects per group, one-way ANOVA, Tukey’s multiple comparison test). C) Histogram summarizing quantitation of total tau (Tau5) immunostaining in Ctrl, AD, and NDAN. Similar to T22, Tau5 expression was also significantly higher in the AD group (p < 0.0001) compared to either Ctrl or NDAN and interestingly, T22 expression was also increased in the NDAN group (p < 0.0001) compared to Ctrl (n = 4 subjects per group, one-way ANOVA, Tukey’s multiple comparison test). D) Representative immunofluorescent images of Ctrl, AD, and NDAN hippocampus showing Tau-5 (green), T22 (red), and merge including DAPI (blue), bar scale: 100μm. E) Histogram summarizing quantitation of tau oligomer (T22) immunostaining in Ctrl, AD, and NDAN. T22 expression was significantly higher in the AD group (p < 0.0001) compared to either Ctrl or NDAN and interestingly, T22 expression was also increased in the NDAN group (p < 0.0001) compared to Ctrl (n = 4 subjects per group, one-way ANOVA, Tukey’s multiple comparison test). F) Histogram summarizing quantitation of total tau (Tau5) immunostaining in Ctrl, AD, and NDAN. Similar to T22, Tau5 expression was also significantly higher in the AD group (p < 0.0001) compared to either Ctrl or (p = 0.0001) NDAN and interestingly, T22 expression was also increased in the NDAN group (**p < 0.0001) compared to Ctrl (n = 4 subjects per group, one-way ANOVA, Tukey’s multiple comparison test).
	Fig. 2. Decreased tau oligomer levels in synaptosomes from the hippocampus and frontal cortex of NDAN subjects. Western blots of crude synaptosomal fractions from six subjects each of control, AD, and NDAN groups obtained from either (A) the frontal cortex or (C) hippocampus were assessed using Tau 5 antibody. Tau oligomeric species identified in the dotted boxed regions (75 kDa to > 250 kDa) were used to quantify and compare between the three groups. Relative oligomeric tau levels detected by Tau 5 antibody (quantified using β-actin loading controls) show significantly lower levels in control and NDAN (B) frontal and (D) hippocampal synaptosomes compared to AD (****p < 0.0003, n = 6, Kruskal-Wallis one-way ANOVA, Dunn’s post-hoc).

	Fig. 4. Differential activation of microtransplanted synaptic AMPA and GABA receptors from the frontal cortex of Ctrl, AD and NDAN subjects. A) GABA currents (elicited using 1 mM GABA), representing the inhibitory component, show no significant differences between Ctrl (clear bars), AD (black bar), or NDAN (grey bar). B) Similar to GABA, Kainate currents (elicited using 100 mM Kainate), representing the excitatory component, show no significant difference between the three groups. Representative ion currents for each group is provided above the bars and represented in normalized units (n.u.) to facilitate comparison of the amplitudes of GABA and kainate currents in the same scale. Kainate, instead of glutamate, was used to avoid AMPA receptors desensitization. C) Linear correlation between maximal kainate and GABA responses measured in single oocytes across groups. Each dot in this figure represents a single recorded oocyte. (r2 are shown next to each group; p < 0.0001). D) Average of the Kainate/GABA ratio in each subject shows reduced values in AD compared to control but not to NDAN. The current ratio was different across groups (with significant reduction between Ctrl and AD (p = 0.0388), but not between Ctrl and NDAN (p > 0.99) or AD and NDAN (p = 0.2268). Each dot represents a single subject. Color schematic for each group are provided in the legends for (C) and (D).
	Fig. 5. Preserved synaptic potentiation in synaptosomes from hippocampus and frontal cortex of NDAN subjects compared to AD patients. Representative density plots showing GluA1-Nrx1β association and detection in different quadrants in size-gated synaptosomes following basal or cLTP treatment in frontal cortex synaptosomes from Ctrl, AD, or NDAN subjects. Fluorescence thresholds (non-specific) were set by staining with secondary antibodies only (lower left quadrant). Percentage GluA1/Nrx1β events increase in the upper right quadrant was used as a measure of cLTP, relative to basal conditions. Percent basal potentiation for Ctrl (clear bars), AD (black filled bars), and NDAN (grey filled bars) observed in synaptosomes from (A) fontal and (B) hippocampal regions are plotted. Synaptosomes from AD frontal cortex (p = 0.0175 compared to Ctrl and p = 0.0283 compared to NDAN, n = 6; one technical repeat, Kruskal-Wallis one-way ANOVA and Dunn’s post-hoc) or hippocampal region (*p = 0.0043 compared to Ctrl and p = 0.0386 compared to NDAN, n = 6; three technical repeats) did not show potentiation above basal levels and was significantly reduced compared to either Ctrl or NDAN group.

References [+] :

Berger, Accumulation of pathological tau species and memory loss in a conditional model of tauopathy. 2007, Pubmed

	Fig. 1. Reduced levels of toxic tau oligomers in hippocampus and frontal cortex of NDAN subjects. A) Representative immunofluorescence images of Ctrl, AD, and NDAN frontal cortex showing Tau5 (green), T22 (red), and merge including DAPI (blue), bar scale: 100μm. B) Histogram summarizing quantitation of tau oligomer (T22) immunostaining in Ctrl, AD, and NDAN. T22 expression was significantly higher in the AD group (**p < 0.0001) compared to either Ctrl or NDAN and interestingly, T22 expression was also increased in the NDAN group (p < 0.0001) compared to Ctrl (n = 4 subjects per group, one-way ANOVA, Tukey’s multiple comparison test). C) Histogram summarizing quantitation of total tau (Tau5) immunostaining in Ctrl, AD, and NDAN. Similar to T22, Tau5 expression was also significantly higher in the AD group (p < 0.0001) compared to either Ctrl or NDAN and interestingly, T22 expression was also increased in the NDAN group (p < 0.0001) compared to Ctrl (n = 4 subjects per group, one-way ANOVA, Tukey’s multiple comparison test). D) Representative immunofluorescent images of Ctrl, AD, and NDAN hippocampus showing Tau-5 (green), T22 (red), and merge including DAPI (blue), bar scale: 100μm. E) Histogram summarizing quantitation of tau oligomer (T22) immunostaining in Ctrl, AD, and NDAN. T22 expression was significantly higher in the AD group (p < 0.0001) compared to either Ctrl or NDAN and interestingly, T22 expression was also increased in the NDAN group (p < 0.0001) compared to Ctrl (n = 4 subjects per group, one-way ANOVA, Tukey’s multiple comparison test). F) Histogram summarizing quantitation of total tau (Tau5) immunostaining in Ctrl, AD, and NDAN. Similar to T22, Tau5 expression was also significantly higher in the AD group (p < 0.0001) compared to either Ctrl or (p = 0.0001) NDAN and interestingly, T22 expression was also increased in the NDAN group (**p < 0.0001) compared to Ctrl (n = 4 subjects per group, one-way ANOVA, Tukey’s multiple comparison test).
	Fig. 2. Decreased tau oligomer levels in synaptosomes from the hippocampus and frontal cortex of NDAN subjects. Western blots of crude synaptosomal fractions from six subjects each of control, AD, and NDAN groups obtained from either (A) the frontal cortex or (C) hippocampus were assessed using Tau 5 antibody. Tau oligomeric species identified in the dotted boxed regions (75 kDa to > 250 kDa) were used to quantify and compare between the three groups. Relative oligomeric tau levels detected by Tau 5 antibody (quantified using β-actin loading controls) show significantly lower levels in control and NDAN (B) frontal and (D) hippocampal synaptosomes compared to AD (****p < 0.0003, n = 6, Kruskal-Wallis one-way ANOVA, Dunn’s post-hoc).

	Fig. 4. Differential activation of microtransplanted synaptic AMPA and GABA receptors from the frontal cortex of Ctrl, AD and NDAN subjects. A) GABA currents (elicited using 1 mM GABA), representing the inhibitory component, show no significant differences between Ctrl (clear bars), AD (black bar), or NDAN (grey bar). B) Similar to GABA, Kainate currents (elicited using 100 mM Kainate), representing the excitatory component, show no significant difference between the three groups. Representative ion currents for each group is provided above the bars and represented in normalized units (n.u.) to facilitate comparison of the amplitudes of GABA and kainate currents in the same scale. Kainate, instead of glutamate, was used to avoid AMPA receptors desensitization. C) Linear correlation between maximal kainate and GABA responses measured in single oocytes across groups. Each dot in this figure represents a single recorded oocyte. (r2 are shown next to each group; p < 0.0001). D) Average of the Kainate/GABA ratio in each subject shows reduced values in AD compared to control but not to NDAN. The current ratio was different across groups (with significant reduction between Ctrl and AD (p = 0.0388), but not between Ctrl and NDAN (p > 0.99) or AD and NDAN (p = 0.2268). Each dot represents a single subject. Color schematic for each group are provided in the legends for (C) and (D).
	Fig. 5. Preserved synaptic potentiation in synaptosomes from hippocampus and frontal cortex of NDAN subjects compared to AD patients. Representative density plots showing GluA1-Nrx1β association and detection in different quadrants in size-gated synaptosomes following basal or cLTP treatment in frontal cortex synaptosomes from Ctrl, AD, or NDAN subjects. Fluorescence thresholds (non-specific) were set by staining with secondary antibodies only (lower left quadrant). Percentage GluA1/Nrx1β events increase in the upper right quadrant was used as a measure of cLTP, relative to basal conditions. Percent basal potentiation for Ctrl (clear bars), AD (black filled bars), and NDAN (grey filled bars) observed in synaptosomes from (A) fontal and (B) hippocampal regions are plotted. Synaptosomes from AD frontal cortex (p = 0.0175 compared to Ctrl and p = 0.0283 compared to NDAN, n = 6; one technical repeat, Kruskal-Wallis one-way ANOVA and Dunn’s post-hoc) or hippocampal region (*p = 0.0043 compared to Ctrl and p = 0.0386 compared to NDAN, n = 6; three technical repeats) did not show potentiation above basal levels and was significantly reduced compared to either Ctrl or NDAN group.