Beyond the brain: early autonomic dysfunction in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is classically defined by central hallmarks such as amyloid-beta plaques, tau hyperphosphorylation, and synaptic failure. However, mounting evidence suggests that dysfunction outside the brain, particularly in the peripheral nervous system, may also play a significant role in disease progression. The adrenal medulla-a key regulator of systemic neurotransmission and stress response-has received little attention in this context. In this study, we investigated whether chromaffin cells (CCs) from the triple transgenic AD mouse model (3xTg) exhibit functional alterations that could contribute to peripheral neurochemical imbalance. Using electrophysiology, high-resolution amperometry, and neurotransmitter quantification, we identified early and progressive defects in CC function. Remarkably, even at two months of age-prior to cognitive decline-3xTg CCs showed impaired exocytosis, reduced vesicle release, and slower fusion pore kinetics. These changes were accompanied by diminished sodium (I_Na), calcium (I_Ca), and nicotinic (I_ACh) currents, compromising CC excitability. With age, a shift toward increased potassium (I_K) currents and enhanced catecholamine secretion may reflect compensatory adaptations aimed at preserving output. These functional deficits were paralleled by structural remodeling of the actin cytoskeleton and systemic neurotransmitter disturbances. Noradrenaline levels increased in both plasma and brain, while dopamine decreased peripherally but paradoxically increased in the prefrontal cortex and hippocampus. Serotonin levels consistently declined across compartments. These imbalances correlated with altered behavior: 3xTg mice displayed increased exploration of exposed areas and heightened behavioral despair, pointing to anxiety- and depression-like phenotypes. Together, our findings identify the adrenal medulla as a previously underrecognized site of early catecholaminergic dysregulation in AD. The observed associations between peripheral CC dysfunction, systemic neurotransmitter imbalance, and behavioral changes point to a potential link between peripheral neuroendocrine alterations and central disease features. These results broaden the current understanding of AD pathophysiology and support the adrenal medulla as a promising candidate for further investigation as a therapeutic target and source of peripheral biomarkers.

Keywords: Alzheimer´s disease; Catecholamine release; Chromaffin cells; Electrophysiology; Neuropsychiatric symptoms; Peripheral dysfunction.

Fig. 1

Vesicle release and exocytosis kinetics are altered in 3xTg chromaffin cells. a Schematic representation of amperometric recordings in cultured chromaffin cells (CC). A carbon fiber electrode is positioned on the surface of a CC (blue) and a voltage of + 700 mV is applied (left). Catecholamines released by the application of an ACh pulse are oxidized and the resulting currents are measured as amperometric spikes (middle). A zoom-in of a representative amperometric spike, highlighting key kinetic parameters, is shown on the right (scale bar: 20 pA; 5 ms). b Representative amperometric recordings in WT (top; scale bar: 50 pA; 10 s) and 3xTg (bottom; scale bar: 100 pA; 10 s) chromaffin cells at 2, 6 and 12 months of age. Cells were stimulated with 100 µM ACh for 1 min (bottom horizontal lines). c Averaged total secretion as the number of amperometric spikes per 1-min recording in CCs of WT and 3xTg mice from different ages. d Averaged total secretion measured as the cumulative charge transfer (Q, pC) from all amperometric spikes per 1-min recording in CCs of WT and 3xTg mice from different ages. e Quantification of rise rate (pA/ms), defined as the slope of the rising phase of amperometric spikes in CCs of WT and 3xTg mice from different ages. f Quantification of Imax (pA), the peak amplitude of amperometric spikes in CCs of WT and 3xTg mice from different ages. g Quantification of the decay time (ms), the time it takes for the current signal to return to baseline after reaching its peak, of the amperometric spikes in CCs of WT and 3xTg mice from different ages. h Quantification of the spike width (t_1/2, ms), the time an event remains above 50% of its peak amplitude, of the amperometric spikes in CCs of WT and 3xTg mice from different ages. Data are presented as mean ± standard error of the mean (SEM). The number of spikes, cells, and mice per group were: 2 m WT (1925, 41, 4); 6 m WT (1152, 33, 4); 12 m WT (1230, 40, 4); 2 m 3xTg (913, 35, 7); 6 m 3xTg (1046, 34, 7); 12 m 3xTg (1234, 30, 5). Statistical significance *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. WT; #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.001 vs. 2 m of the same genotype

Fig. 2

Ion channel currents in the chromaffin cell activation cycle are altered in 3xTg mice. a Scheme of the experimental approach: a recording pipette was placed in a chromaffin cell (blue), and a puffing pipette was located nearby. b Representative nicotinic acetylcholine receptor (nAChR) current with a 250 ms ACh pulse from WT and 3xTg mice at the different ages studied (scale bar: 250 pA; 500 ms). c Averaged nAChR current (I_ACh) in CCs of WT and 3xTg mice from different ages. d Top left: Voltage-clamp protocol for voltage-gated sodium channels. Square depolarizing 10 ms pulses were applied from − 60 to + 60 mV in 10 mV increments from a holding potential of − 80 mV at 15 s intervals. Average current–voltage (I–V) relationship for voltage-gated sodium channels in CCs from WT and 3xTg mice at different ages. e Top: Representative peak sodium currents (I_Na) at − 30 mV from WT and 3xTg mice at the different ages studied (scale bar: 500 pA; 5 ms). Bottom: Averaged peak I_Na in CCs of WT and 3xTg mice from different ages. I_Na peak was usually reached at approximately − 30 mV. f Top left: Voltage-clamp protocol for voltage-gated calcium channels. Square depolarizing 50 ms pulses were applied from − 50 to + 50 mV in 10 mV increments from a holding potential of − 80 mV at 10 s intervals. Average I–V relationship for voltage-gated calcium channels in CCs from WT and 3xTg mice at different ages. g Top: Representative peak calcium currents (I_Ca) at 0 mV from WT and 3xTg mice at the different ages studied (scale bar: 100 pA; 25 ms). Bottom: Averaged peak I_Ca in CCs of WT and 3xTg mice from different ages. I_Ca peak was usually reached at 0 mV. h Top left: Voltage-clamp protocol for calcium-dependent potassium channels. Square depolarizing 400 ms pulses were applied from − 40 to + 150 mV in 10 mV increments from a holding potential of − 80 mV at 20 s intervals. To isolate the calcium-dependent component of these currents, an external solution containing 2 mM Ca²⁺ was applied (2 Ca²⁺). Average I-V relationship for calcium-dependent potassium channels in CCs from WT and 3xTg mice at different ages. i Top: Representative peak calcium-dependent potassium currents (I_K(Ca)) at + 60 mV from WT and 3xTg mice at the different ages studied (scale bar: 2 nA; 100 ms). Bottom: Averaged peak I_K(Ca) in CCs of WT and 3xTg mice from different ages. I_K(Ca) peak was usually reached at + 60 mV. j Top left: Voltage-clamp protocol for voltage-dependent potassium channels. Square depolarizing 400 ms pulses were applied from − 40 to + 150 mV in 10 mV increments from a holding potential of − 80 mV at 20 s intervals. To eliminate the calcium-dependent component, an external solution with 0 mM Ca²⁺ was applied (0 Ca²⁺). Average I–V relationship for voltage-dependent potassium channels in CCs from WT and 3xTg mice at different ages. k Top: Representative peak voltage-dependent potassium currents (I_K(V)) at + 150 mV from WT and 3xTg mice at the different ages studied (scale bar: 2 nA; 100 ms). Bottom: Averaged peak I_K(V) in CCs of WT and 3xTg mice from different ages. I_K(V) peak was reached at + 150 mV. Data are presented as mean ± SEM. Each dot represents a different cell from at least three different mice. Statistical significance *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. WT; #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.001 vs. 2 m of the same genotype

Fig. 3

Early alterations in chromaffin cell excitability in 3xTg mice are compensated over time. a Representative resting membrane potential recordings in WT (top) and 3xTg (bottom) chromaffin cells at 2, 6 and 12 months of age (scale bar: 60 s). The dashed line represents each genotype's resting membrane potential (RMP) at 2 m. b Averaged RMP in CCs of WT and 3xTg mice from different ages. c Averaged spontaneous action potential firing (sAP) in CCs of WT and 3xTg mice from different ages. d Representative membrane potential recordings in WT (top) and 3xTg (bottom) chromaffin cells at 2, 6 and 12 months of age with a 1-min ACh (100 µM) pulse (scale bar: 10 s). The dashed line represents each genotype's ACh evoked depolarization at 2 m. e Averaged membrane potential depolarization from its resting state to the maximum depolarization after ACh was applied in CCs of WT and 3xTg mice from different ages. f Averaged action potential firing evoked during the 1-min pulse of ACh (eAP) in CCs of WT and 3xTg mice from different ages. Data are presented as mean ± SEM. Each dot represents a different cell from at least three different mice. Statistical significance *p ≤ 0.05; **p ≤ 0.01 vs. WT; ##p ≤ 0.01 vs. 2 m of the same genotype

Fig. 4

Progressive disruption of the actin cytoskeleton in 3xTg chromaffin cells. a Scheme of the experimental approach: chromaffin cells were stained to visualize the actin cytoskeleton (red filaments) using Phalloidin 546 and nuclei (blue) with DAPI. b Pseudocolor confocal images showing WT CCs (top) and 3xTg CCs (bottom) at different ages stained with Phalloidin 546 to label F-actin (red) and DAPI to label the nucleus (blue) (scale bar: 5 µm). c Left. Averaged F-actin intensity was measured in arbitrary units (AU) in CCs of WT and 3xTg mice of different ages. Right. Averaged cortical/cytosolic phalloidin intensity ratio in CCs of WT and 3xTg mice from different ages. Data are presented as mean ± SEM. Each dot represents a different cell from 4 different mice for each genotype and age. Statistical significance *p ≤ 0.05; ***p ≤ 0.001 vs. WT; ###p ≤ 0.001 vs. 2 m of the same genotype

Fig. 5

Altered plasma catecholamine and serotonin levels in 3xTg mice during Alzheimer’s disease progression. a Schematic representation of the experimental approach: blood was collected from WT and 3xTg mice. After plasma separation, neurotransmitter levels were measured using high-performance liquid chromatography-mass spectrometry (HPLC–MS). b Adrenaline levels in plasma from WT and 3xTg mice at different ages. c Noradrenaline levels in plasma from WT and 3xTg mice at different ages. d Dopamine levels in plasma from WT and 3xTg mice at different ages. e Serotonin levels in plasma from WT and 3xTg mice at different ages. Data are presented as mean ± SEM. Each dot represents an individual plasma sample, with at least three different mice per genotype and age group. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. WT; #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.001 vs. 2-month-old mice of the same genotype

Fig. 6

Altered anxiety- and depressive-like behaviors in 3xTg mice during Alzheimer’s disease progression. a Schematic representation of the open field test (OFT), used to assess anxiety-like behavior by measuring thigmotactic behavior and exploration tendencies. b Representative tracking plots (left) and heat maps (right) of a 12-month-old (12 m) WT and 3xTg mice in the OFT. c Quantification of the total distance traveled (cm) in the inner zone of the open field arena across different ages. d Distance traveled (cm) along the walls of the open field. e Time spent (s) in the inner zone of the arena. f Number of entries into the inner zone. g Schematic representation of the elevated plus maze (EPM), consisting of two open and two closed arms, used to assess anxiety-related behavior. h Time spent in the open arms of the elevated plus maze by 12 m WT and 3xTg mice. i Time spent in the closed arms of the maze for 12 m WT and 3xTg mice. j Time spent at the edges of the open arms, an indicator of increased exploratory behavior and potential disinhibition of fear responses. k Schematic of the tail suspension test (TST), used to evaluate depressive-like behavior by measuring immobility time. l Percentage of immobility time in the TST across different ages. Data are presented as mean ± SEM. Each dot represents an individual mouse. Statistical significance: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 vs. WT; #p ≤ 0.05 vs. 2 m of the same genotype