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The Unexpected Role of NaV1.6 Channels in Alzheimer’s Disease

By Alomone Team • November 2023

A lot of Alzheimer’s disease (AD) research centers around amyloid-beta (Aβ) and its accumulation. Less work has linked Aβ and its interaction with ion channels. Interesting research, published in Scientific Reports, has uncovered a crucial link between a specific type of voltage-gated sodium (NaV) channel and the hyperexcitability of neurons in AD. Not only does this research give new insight into the role of NaV channels, but opens up a promising avenue for potential therapeutic interventions that could counteract the early stages of AD.

How does Aβ relate to NaV1.6?


In the search for answers, researchers turned their attention to voltage-gated sodium channels, which are essential for regulating neuronal excitability. These channels have been implicated in the hyperactivity of hippocampal neurons in AD and the increased incidence of seizures. The team embarked on a journey, using primary hippocampal neurons exposed to amyloid-β1–42 (Aβ1–42) oligomers, a hallmark of AD, to unravel the mysteries of neuronal hyperexcitability. The team relied on two different in vitro models to recapitulate amyloid pathology: primary cultures of hippocampal neurons from mice treated with syn-thetic Aβ1–42 oligomers for 24 hours, and primary cultures of hippocampal neurons from Tg2576 mice, which endogenously produce Aβ1–42 peptides that accumulate over time in culture. They also used the sodium channel blocker Tetrodotoxin citrate (TTX) (#T-550) to block endogenous NaV currents.

After exposing hippocampal neurons to Aβ1–42 western blot with anti-NaV1.6(SCN8A) antibody (#ASC-009) showed an accumulation of these Aβ1–42 oligomers after 24 hours and selectively upregulated the expression and activity of NaV1.6 channels (Figure 1). The team also used Anti-SCN1A (NaV1.1) antibody (#ASC-001) and Anti-SCA2A (NaV1.2) antibody (#ASC-002) to look at the expression of those proteins in response to Aβ1–42. They found that the selective upregulation of NaV1.6 played a crucial role in membrane depolarization and the increase in spike frequency – the culprit behind neuronal

To confirm their findings, the researchers turned to Tg2576 mouse embryos, a common mouse model for studying Alzheimer’s disease. They discovered that hippocampal neurons obtained from these mice also displayed the selective upregulation of NaV1.6 channels, with the same lack of effect on NaV1.1 and NaV1.2 channels, as well as increased spike frequency, membrane depolarization, and current density.

These findings provided the necessary link between Aβ1–42 oligomers and the NaV1.6 channels, solidifying the channel’s role in the hyperexcitability of hippocampal neurons.

 

Aβ1–42 exposure selectively upregulates NaV1.6 protein expression and activity in primary hippocampal neurons

Figure 1. Effect of Aβ1–42 exposure on Na+ currents in primary hippocampal neurons at 10–12 DIV. (A) Representative traces of Na+ currents recorded in primary hippocampal neurons under control conditions and after 24 h of 0.1 μM, 1 μM and 5 μM Aβ1–42. (B) Normalization of Na+ current densities at −20 mV recorded from primary hippocampal neurons under control conditions and after 24 h of 0.1 μM, 1 μM, 5 μM Aβ1–42, and 5 μM Aβ42-1. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. *p < 0.05 versus control, ***p < 0.001 versus control, ^p < 0.05 versus 0.1 μM, #p < 0.001 versus 0.1 and 1 μM. (C) Normalization of Na+ current densities at −20 mV recorded from primary hippocampal neurons under control conditions and after 1 h, 12 h, 24 h, and 48 h of 5 μM Aβ1–42. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ***p < 0.001 versus control, ^p < 0.001 versus 1 h, #p < 0.001 versus 1 h and 12 h, §p < 0.001 versus 24 h. (D) Representative current tracings recorded in the gap-free mode under control conditions and after 5 μM Aβ1–42 (24 h) in primary hippocampal neurons. (E) Quantification of Aβ1–42 effects on spike frequency under control conditions and after 5 μM Aβ1–42 (24 h) in primary hippocampal neurons. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ***p < 0.001 versus control. (F) Quantification of Aβ1–42 effects on membrane potential under control conditions and after 5 μM Aβ1–42 (24 h) in primary hippocampal neurons. The number of cells used for each experimental condition is noted on the bars, values are expressed as percentage mean ± SEM of 3 independent experimental sessions. ***p < 0.001 versus control. From Ciccone, R. et al. Sci Rep9, 13592 (2019).

Expressing and silencing NaV1.6

Now that they’d uncovered the connection between Aβ1–42 and NaV1.6, the researchers delved deeper into the mechanisms at work and explored what role NaV1.6 was playing in Aβ1–42-induced neuronal excitability. To do this, they used NaV1.6 siRNA and anisomycin treatment in an attempt to revert the electrophysiology changes.

Both of these approaches significantly reduced Na+ currents, prevented electrophysiological changes (including the increase of spike frequency and membrane depolarization), and even reduced the amplitude and frequency of spontaneous action potentials in the Tg2576 mice hippocampus.

In an attempt to explore the expression in a little more detail in their mouse model, the team double-labeled tissue neurons with anti-NaV1.6 channels (#ASC-009) and anti-microtubule-associated protein (MAP2). Here they saw punctuated NaV1.6 staining throughout the neuropil and soma of WT hippocampal neurons (Figure 2A) and pronounced perikaryal staining in Tg2576 hippocampal neurons (Fig 2A d–f) when compared to WT neurons (Figure 2A a–c). They also saw how anisomycin treatment reversed the increase in NaV1.6 immunoreactivity in Tg2576 hippocampal neurons (Figure 2A g–i).

These results strongly supported the specific involvement of NaV1.6 channels in Aβ1–42-induced neuronal hyperexcitability.

Anisomycin treatment reduced the increase in NaV1.6 immunoreactivity in Tg2576 hippocampal neurons

Figure 2. Immunocytochemical analysis of NaV1.6 (Anti-Nav1.6(SCN8A) #ASC-009) protein expression after anisomycin treatment in Tg2576 primary hippocampal neurons at 12 DIV. (A) Confocal double immunofluorescence images displaying NaV1.6 (green) and MAP2 (red) distribution in WT (a-c) and Tg2576 primary hippocampal neurons in the absence (d-f) or in the presence (g-i) of anisomycin. Scale bars in a-i: 20 μm. (B) Quantitative analyses of NaV1.6-positive puncta within the soma of WT and Tg2576 primary hippocampal neurons in the absence or in the presence of anisomycin. Scale bars: 5 μm. Data are expressed as mean ± SEM of values obtained from 20 cells per group in 3 independent experimental sessions. **p < 0.01 versus WT; #p < 0.001 versus Tg2576. From Ciccone, R. et al. Sci Rep9, 13592 (2019).

Solving the mystery

With the puzzle pieces falling into place, the researchers began to unravel the mystery of how Aβ1–42 oligomers increased NaV1.6 activity in hippocampal neurons. They discovered that Aβ1–42 oligomers, along with intracellular accumulation, led to a selective increase in NaV1.6 protein expression and functional activity. Interestingly, this upregulation was also observed in Tg2576 hippocampal neurons, reinforcing the connection between Aβ1–42 and NaV1.6 channels.

While the exact mechanisms by which Aβ1–42 oligomers increase NaV1.6 activity are still unclear, the researchers speculate that it may involve altered gene transcription, reduced protein degradation, or the interaction of Aβ1–42 with other proteins such as the amyloid precursor protein (APP), which can influence the translocation of NaV1.6 channels to the cell membrane.

Unanswered Questions around NaV1.6 and AD

While the road beyond the research is interesting, there are still unanswered questions and challenges to overcome. The researchers have provided compelling evidence linking NaV1.6 to neuronal hyperexcitability in the context of AD. However, the precise mechanisms by which Aβ1–42 oligomers increase NaV1.6 activity remain unknown. Understanding these mechanisms will be crucial for the development of targeted therapies.

One intriguing aspect is the interaction between Aβ1–42 oligomers and the ubiquitin system, which is responsible for protein degradation. It has been observed that Aβ1–42 oligomers can disrupt the ubiquitin system, leading to decreased protein degradation in AD mouse models. Could this dysregulation contribute to the upregulation of NaV1.6? Further investigations into the intricate relationship between Aβ1–42 and the ubiquitin system may reveal additional therapeutic targets.

While this study focused on its involvement in neuronal hyperexcitability, NaV1.6 may have broader implications. As we continue to unravel the complexities of AD, it is crucial to investigate whether NaV1.6 contributes to other disease-related processes, such as synaptic dysfunction and neuroinflammation. Such knowledge could potentially unveil new avenues for intervention.

NaV1.6: A Significant New Player

This research not only sheds light on the role of NaV1.6 channels in AD but also offered potential therapeutic avenues. By targeting the upregulation and hyperactivity of NaV1.6 channels, researchers could theoretically counteract the early stages of hippocampal hyperexcitability and subsequent cognitive deficits in AD by targeting the selective upregulation of NaV1.6 channels.

It’s always important to remember that translating these findings into clinical applications will be a formidable task. Developing drugs that specifically target NaV1.6 while avoiding off-target effects is a challenge that drug discovery scientists around the world continue to tackle. Nevertheless, the potential rewards are immense.

The implications of this research extend beyond AD. Hyperexcitability and disrupted neuronal networks are features seen in other neurological conditions, such as epilepsy. Understanding the role of NaV1.6 in these disorders could have broader implications for the field of neuroscience.

In summary, this research brings us one step closer to understanding the intricate workings of the brain in AD. By uncovering the pivotal role of NaV1.6 in neuronal hyperexcitability induced by Aβ1–42 oligomers, scientists here have provided a promising target for future therapies. While there is still much work to be done, these findings pave the way for a potential breakthrough in the treatment of AD and other neurological disorders as well as putting ion channels firmly in the research spotlight.

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