Neuronal α-amylase is important for neuronal activity and glycogenolysis and reduces in presence of amyloid beta pathology

Abstract

Recent studies indicate a crucial role for neuronal glycogen storage and degradation in memory formation. We have previously identified alpha-amylase (α-amylase), a glycogen degradation enzyme, located within synaptic-like structures in CA1 pyramidal neurons and shown that individuals with a high copy number variation of α-amylase perform better on the episodic memory test. We reported that neuronal α-amylase was absent in patients with Alzheimer's disease (AD) and that this loss corresponded to increased AD pathology. In the current study, we verified these findings in a larger patient cohort and determined a similar reduction in α-amylase immunoreactivity in the molecular layer of hippocampus in AD patients. Next, we demonstrated reduced α-amylase concentrations in oligomer amyloid beta 42 (Aβ₄₂ ) stimulated SH-SY5Y cells and neurons derived from human-induced pluripotent stem cells (hiPSC) with PSEN1 mutation. Reduction of α-amylase production and activity, induced by siRNA and α-amylase inhibitor Tendamistat, respectively, was further shown to enhance glycogen load in SH-SY5Y cells. Both oligomer Aβ₄₂ stimulated SH-SY5Y cells and hiPSC neurons with PSEN1 mutation showed, however, reduced load of glycogen. Finally, we demonstrate the presence of α-amylase within synapses of isolated primary neurons and show that inhibition of α-amylase activity with Tendamistat alters neuronal activity measured by calcium imaging. In view of these findings, we hypothesize that α-amylase has a glycogen degrading function within synapses, potentially important in memory formation. Hence, a loss of α-amylase, which can be induced by Aβ pathology, may in part underlie the disrupted memory formation seen in AD patients.

Keywords: Alzheimer's disease; alpha-amylases; amyloid beta-peptides; calcium imaging; glycogen; induced pluripotent stem cells; tendamistat.

FIGURE 1

Immunohistochemical staining of alpha (α)‐amylase of human hippocampal postmortem tissue. Image in (a) shows an α‐amylase staining of Cornius ammonis 1 (CA1) and the molecular layer (ML) in a non‐demented control (NC). Higher magnification of the CA1 region in (b) shows that the α‐amylase immunoreactivity (IR) is found in dendritic spine‐like structures (DS). Higher magnification of the ML region in (c) shows that α‐amylase IR in ML forms a grained pattern. Image in (d) represent an α‐amylase staining of CA1 and ML in a patient with Alzheimer's disease (AD). Higher magnification of the CA1 region in (e) shows α‐amylase IR in form of Hirano bodies, which is also seen in the ML region in (f). Graph in (g and h) shows that scores of the DS α‐amylase IR in CA1 (g) and scores of grained α‐amylase IR in ML (h) are significantly higher in NC compared to AD. Scatterplot in (i–j) demonstrates a negative correlation between DS α‐amylase IR in CA1 and amyloid beta (Aβ) ABC scores (i) and neurofibrillary tangles (NFT) Braak scores in (j). Scale bar in (a and d) = 50 μm, in b–c = 20 μm and (e–f) = 10 μm. Graphs in (g–h) is presented as mean ± SD, and statistical analysis was done using t test. The correlation analyses in (i–j) were done using Spearman correlation test. ***indicates p‐value < 0.001, **indicates p‐value < 0.01

FIGURE 2

Alpha‐amylase in hiPSC‐derived neurons and oligomer amyloid beta‐42 stimulated SHSY5Y cells. Image in (a) shows hiPSC‐derived neurons without mutation in PSEN1 gene (isogenic control) (L150P‐GC) immunoflourescently stained against salivary α‐amylase (green) with DAPI marker nuclei (magenta). A hiPSC neuronal process is shown in higher magnification in (b) where the arrow indicates α‐amylase (green) and phalloidin (magenta) in close association. Graph in (c) shows the significant lower levels of α‐amylase in L150P compared to L150P‐GC. Image in (e) shows control (ctrl) SH‐SY5Y cells immunofluorescent staining of α‐amylase (green), and nuclei are visualized with DAPI (magenta). The graph in (f) shows significantly lower α‐amylase concentrations in amyloid beta 42 oligomers Aβ42O stimulated cells compared to Ctrl. Scale bar in (a and e) indicates 15 μm, and scale bar in (b) indicates 0.5 μm. Statistical analysis was done with t test, and values in graphs (c and f) are presented as mean ± SD. ***indicates p‐value < 0.001, **indicates p‐value < 0.0, *p‐value < 0.05

FIGURE 3

Glycogen in siRNA transfected SH‐SY5Y cells, Tendamistat or amyloid beta‐42 stimulated SH‐SY5Y cells and hiPSC‐derived neurons. (a–c) show representative images of SHSY5Y cells immunostained against glycogen (magenta) and cell nuclei marker DAPI (green) after being transfected with α‐amylase siRNA (siAMY) and non‐targeting siRNA (siCtrl) (a), stimulated with Tendamistat for 2 h (Tenda 2 h) and vehicle control (Ctrl) (b), and stimulated with Amyloid beta 42 oligomers (Aβ42O) and vehicle control (Ctrl) (c). Image in (d) shows glycogen (magenta) and DAPI (green) in neurons derived from hiPSC with mutation in PSEN1 (L150P) and isogenic control (L150P‐GC). Scale bar indicates 15 μm. Graph in (e) shows significant larger glycogen area/cell in SH‐SY5Y cells transfected with siAMY compared to siCtrl). Graph in (b) demonstrates significantly larger glycogen area/cell in SH‐SY5Y cells stimulated with Tenda. 2 h compared compared to Ctrl. Graph in (c) shows significantly smaller glycogen area/cell in Aβ42O stimulated SH‐SY5Y cells compared to Ctrl. Graph in (d) demonstrates significantly smaller glycogen area /cell in L150P compared L150P‐GC. Statistical analysis was done using t test, and data are presented as mean ± SD, ***indicates p‐value < 0.001, **indicated p‐value < 0.01, and *indicates p‐value < 0.05

FIGURE 4

Cellular localization of alpha (α)‐amylase in primary mouse neurons. Image in (a) shows immunofluorescent staining of α‐amylase with antibody directed against salivary α‐amylase (green) and neuronal marker microtubule‐associated protein 2 (MAP2) (magenta). The α‐amylase immunoreactivity is seen in the cell body (arrowhead) and along the dendrites (arrow). Confocal image in (b) shows a primary mouse neuronal process where α‐amylase staining (green) and phalloidin (magenta) are in close association. Confocal image in (c) shows immunofluorescent staining of primary mouse neuronal process where α‐amylase (green) and synaptotagmin (magenta) are in close association (arrow). Confocal image in (d) shows a close association between α‐amylase (green) and CAMKII (magenta) (arrow). Confocal image in (e) shows a primary mouse neuron stained against α‐amylase (green) and glycogen (magenta). The white squares indicate magnification of the area seen in (f and g). Arrows in (f‐g) indicate an close association between α‐amylase and glycogen. Scale bar in (a and e) indicates 5 μm, scale bar in (d and g) indicates 0.5 μm

FIGURE 5

Calcium imaging of Tendamistat treated primary mouse neurons. Micrograph in (a) shows maximum intensity projections of the imaging time series of primary neuron culture labeled with Fluo4 AM. Scale bar in a indicates 100 μm. Graph in (b) shows frequency of spikes per minutes in primary mouse neurons stimulated with α‐amylase inhibitor Tendamistat for 2 h (Tenda. 2 h). Graph in (c) demonstrates significantly lower amplitude of calcium concentrations (spike height) after 2 h stimulation with Tendamistat compared to vehicle control (Ctrl). Graph in (c) demonstrates significantly shorter inter‐spike intervals after 2 h Tendamistat stimulation compared to Ctrl. Statistical analysis was performed by Mann–Whitney U test, and data are presented as median and range ***indicates p‐value < 0.001 and *indicates p‐value < 0.05