Dose-dependent phosphorylation of endogenous Tau by intermittent hypoxia in rat brain

Abstract

Intermittent hypoxia, or intermittent low oxygen interspersed with normal oxygen levels, has differential effects that depend on the "dose" of hypoxic episodes (duration, severity, number per day, and number of days). Whereas "low dose" daily acute intermittent hypoxia (dAIH) elicits neuroprotection and neuroplasticity, "high dose" chronic intermittent hypoxia (CIH) similar to that experienced during sleep apnea elicits neuropathology. Sleep apnea is comorbid in >50% of patients with Alzheimer's disease-a progressive, neurodegenerative disease associated with brain amyloid and chronic Tau dysregulation (pathology). Although patients with sleep apnea present with higher Tau levels, it is unknown if sleep apnea through attendant CIH contributes to onset of Tau pathology. We hypothesized CIH characteristic of moderate sleep apnea would increase dysregulation of phosphorylated Tau (phospho-Tau) species in Sprague-Dawley rat hippocampus and prefrontal cortex. Conversely, we hypothesized that dAIH, a promising neurotherapeutic, has minimal impact on Tau phosphorylation. We report a dose-dependent intermittent hypoxia effect, with region-specific increases in 1) phospho-Tau species associated with human Tauopathies in the soluble form and 2) accumulated phospho-Tau in the insoluble fraction. The latter observation was particularly evident with higher CIH intensities. This important and novel finding is consistent with the idea that sleep apnea and attendant CIH have the potential to accelerate the progression of Alzheimer's disease and/or other Tauopathies.NEW & NOTEWORTHY Sleep apnea is highly prevalent in people with Alzheimer's disease, suggesting the potential to accelerate disease onset and/or progression. These studies demonstrate that intermittent hypoxia (IH) induces dose-dependent, region-specific Tau phosphorylation, and are the first to indicate that higher IH "doses" elicit both endogenous, (rat) Tau hyperphosphorylation and accumulation in the hippocampus. These findings are essential for development and implementation of new treatment strategies that minimize sleep apnea and its adverse impact on neurodegenerative diseases.

Keywords: Alzheimer’s disease; Tau phosphorylation; intermittent hypoxia; sleep apnea.

Graphical abstract

Figure 1.

Experimental timeline for different intermittent hypoxia protocols. Rats were acclimatized for 14 days (14 D) prior to beginning 7 (7 D IH) or 28 days of intermittent hypoxia (28 D IH). On the 8th day (8 D) or 29th day (29 D), rats were anesthetized and hippocampal and prefrontal cortex were harvested (A). Normoxia (NX) consisted of 21% O₂ for 8 h/day (B). Daily acute IH (dAIH) consisted of 10, 5-min episodes of 10.5% O₂ alternating with 5-min normoxic intervals for a total duration of 1.5 h/day (C). Mild chronic IH (CIH5/5) consisted of 5-min episodes of 10.5% O₂ alternating with 5-min normoxic intervals for 8 h/day (D). Moderate CIH (CIH2/2) consisted of 2-min episodes of 10.5% O₂ alternating with 2-min normoxic intervals (E).

Figure 2.

Intermittent hypoxia increases Tau phosphorylation in rat hippocampus. Representative Western blots of phospho- (PHF-1 and CP13) and total Tau (Tau5) after 7 (left column; 7 D IH) or 28 (right column; 28 D IH) days of normoxia (NX7; NX28), daily AIH (dAIH7; dAIH28), mild (CIH7:5/5; CIH28:5/5), or moderate (CIH7:2/2; CIH28:2/2) CIH (A). Densitometric quantification of PHF-1 and CP13 normalized to Tau5 (B). Soluble phospho-Tau levels were significantly increased with CIH7:2/2, and in all 28 D groups (dAIH28, CIH28:5/5, and CIH28:2/2). Data are reported as means ± SE; n = 6–8/group; *P < 0.050.

Figure 3.

Intermittent hypoxia increases Tau phosphorylation in rat prefrontal cortex after 28 days. Representative Western blot of phospho-Tau (PHF-1 and CP13) and total Tau (Tau5) after 7 (left column; 7 D IH) or 28 (right column; 28 D IH) days of normoxia (NX7 or NX28), daily AIH (dAIH7 or dAIH28), mild (CIH7:5/5 or CIH28:5/5), or moderate (CIH7:2/2 or CIH28:2/2) CIH (A). Densitometric quantification of PHF-1 and CP13 normalized to Tau5 (B). Soluble phospho-Tau levels were significantly increased only in groups exposed to CIH for 28 days (dAIH28, CIH28:5/5, and CIH28:2/2). No differences in soluble phospho-Tau were detected in any group exposed to 7 D IH. Each circle is an individual rat. Data are reported as means ± SE; n = 6 or 7/group. *P < 0.050.

Figure 4.

Mild and moderate chronic intermittent hypoxia for 28 days increases hippocampal accumulation of insoluble Tau. Representative Western blot of phospho-Tau (PHF-1 and CP13) and total Tau (Tau5) after 28 days (right column; 28 D IH) of normoxia (NX28), daily AIH (dAIH28), mild (CIH28:5/5), or moderate (CIH28:2/2) CIH (A). Densitometric quantification of PHF-1 and CP13 normalized to Tau5 (B). Insoluble phospho-Tau was significantly increased in hippocampal CIH28:5/5 and CIH28:2/2 fractions, only. Data are reported as means ± SE; n = 5 or 6/group; *P < 0.050.