Helu Zhang
Department of Psychology, Monash University, Melbourne, Australia.
DOI: 10.4236/aar.2025.142005   PDF    HTML   XML   138 Downloads   1,248 Views   Citations

Abstract

Alzheimer’s disease (AD) is highly prevalent in the elderly population and leads to AD patients’ higher mortality, low life quality, and lead to a huge economic burden on the health system. Even though the APOEε4 gene has been identified as a risk factor for the late onset of AD, there are no studies to examine the impact of APOEε4 on the neural and gene expression mechanisms of cognitive impairment in AD. Our study examined the impact of APOEε4 on AD patients’ cognitive function and the level of a hallmark of AD pathology. This study also examined the impact of APOEε4 on the number of neurons in the dorsolateral prefrontal cortex (DLPFC)and the gene expression of degenerating neurons. This study used data from one publicly available dataset called the Seattle Alzheimer’s Disease Brain Cell Atlas consortium (SEA-AD), including 75 AD patients (M = 88.56 years, SD = 7.89). T-tests revealed a significant difference in participants’ age at death, cognitive status, age of onset cognitive symptoms, cognitive abilities screening instrument score, mini-mental state examination score, montreal cognitive assessment score, and the percentage of Sst chodl, L6 b, and L5/6 NP cells between APOEε4 carriers and non-carriers. Single-cell RNA sequence revealed that APOEε4 led to a significantly less gene expression of the GLRA1 gene in Sst chodl neurons and KCNA1 gene in L5/6 NP neurons. The present findings provide insight for enhancing understanding of the cause of AD and AD’s cognitive impairment from an APOEε4 perspective.

Keywords

Alzheimer’s Disease, APOEε4, Cognitive Impairment, Differential Gene Expression, Single-Cell RNA Sequence

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1. Introduction

Alzheimer’s disease (AD) is a progressive neurogenerative disorder and is related to the most common cause of dementia, accounting for about 60% - 80% of these cases [1]. It is estimated that nearly 1 million new cases are diagnosed each year [2]. The treatment of AD imposes a huge burden on the health system, with nearly $600 million per year [3]. AD is characterized by memory loss and cognitive impairment, which reduce the elderly population’s normal daily function, increase dependence on people, and even impact the mortality of the elderly population [4] [5]. Nearly 33 % of the elderly population over 65 years old died due to suffering from AD and dementia [6]. Pathologically, the hallmarks and risk factors related to AD include the increase of amyloid plaques caused by the accumulation of amyloid-β (Aβ) peptide, the increase of neurofibrillary tangles (NFTs) caused by the deposition of phosphorylated tau, as well as synaptic dysfunction, inflammation [7]-[9].

Genetic risk factors impact these pathogenic pathways related to late-onset AD (LOAD) and thus increase the risk for AD [10]. The strongest risk factor for LOAD is apolipoprotein E (apoE), with three predominant APOE alleles [11]. Among these three predominant APOE alleles, ε4 (APOEε4) alleles are considered a significant risk factor for developing AD [12] [13]. People with APOEε4 experience a higher risk of suffering from dementia and poorer cognitive function [14]-[16]. Individuals with APOEε4 show earlier accumulation of Aβ and increased Aβ levels than noncarriers [17]-[20]. The frequency of APOEε4 is also significantly higher in AD patients than in healthy populations [21]. Additionally, APOEε4 compromises Aβ clearance by disrupting receptor-mediated clearance and proteolytic degradation [22] [23]. This results in increased Aβ deposition in APOEε4 carriers, leading to synaptic dysfunction, synaptic loss, cognitive impairment, and a higher risk of AD [24]-[26]. In addition, APOEε4 carries show increased phosphorylated tau deposition compared to noncarriers, regardless of the presence of Aβ [27], which further exacerbates synaptic dysfunction and neurodegeneration, leading to a higher risk of AD [28] [29].

APOEε4 is associated with neurodegeneration and interruption of the normal neuron function, contributing to AD. APOEε4 promotes Aβ accumulation [17]-[20], which could interfere with ion channels critical for synaptic transmission [30]-[33]. Dysfunctional ion channels may lead to synapse loss in AD [34]-[36]. Thus, APOEε4 may be one reason leading to the number of neurons declining in AD patients and cognitive impairment [37] [38]. However, there is no direct evidence to show the impact of APOEε4 on the number of neurons and on the gene expression in specific neurons.

Given the high mortality and prevalence, low life quality, and huge economic burden related to AD [3]-[6], understanding gene factors related to AD is important to enhance the understanding of the mechanisms of underlying the cause of AD. Despite literature suggesting APOEε4 is related to AD and increases the accumulation of Aβ, little is known about the impact of APOEε4 on neuron neurodegeneration and whether APOEε4 influences gene expression in these neurons. This current study aims to explore the impact of APOEε4 on cognitive function, the level of hallmarks of pathology related to AD, the number of neurons, and gene expression related to these neurons. Hypothesis 1: Compared with APOEε4 non-carriers, APOEε4 carriers will have a significantly younger age at death, younger age of onset AD, younger age of dementia diagnosis, and lower score in cognitive function test. Hypothesis 2: Compared with APOEε4 non-carriers, APOEε4 carriers will have a significantly decreased number of neurons in the prefrontal cortex.

2. Materials and Method

This study used the Seattle Alzheimer’s disease Brain Cell Atlas consortium (SEA-AD) [39], which is a publicly available dataset, including 85 participants’ information (including 75 AD patients and 10 healthy participants) on AD pathology, cognitive condition, whether are APOEε4 carriers, cell types in the dorsolateral prefrontal cortex (DLPFC), and single-nucleus RNA sequence. This study used 75 AD patients’ information about AD pathology, AD patients’ cognitive condition, AD’s life span, and AD patients’ APOEε4 information (APOEε4 carriers and APOEε4 non-carriers) to investigate the impact of APOEε4 on AD’s cognitive function and pathology. Then, this study used AD patients’ information about the number of neurons in the DLPFC, a brain region related to working memory and executive function [40], to explore the role of APOEε4 in different neurons. To investigate the impact of APOEε4 on gene expression of specific cell types, the current study used a single-nucleus RNA sequence.

3. Bioinformatics Tools

The calculation of the percentage of different cell types in DLPFC was done in Python (3.12.2). The statistics tests on the impact of APOEε4 on various factors were done in R studio (v 4.4.2). In addition, the human motor cortex is used as the reference to analyze the single-cell RNA sequence by using the Seurat toolbox in R [41].

4. Results

4.1. Descriptive Statistics

Participant characteristics are described in Table 1. A total of N = 75 participants were included in this study, of which 46 were female with an average age at death of 89, and 29 were male with an average age at death of 88.

4.2. The Impact of APOEε4 Gene on AD Patient’s Cognitive Function

Several t-tests were conducted to examine whether there was a significant difference between APOEε4 carriers and non-carriers in participants’ age at death, cognitive status, age of onset cognitive symptoms, cognitive abilities screening instrument score, mini-mental state examination score, and montreal cognitive assessment score. The significant difference between APOEε4 carriers and non-carriers that have significant P values (<0.05) and effect sizes (Cohen’s d) is shown in Figure 1 and Table 2.

Figure 1. The P-value of the Effect of the APOEε4 on Cognitive Function. Note: From (a)-(e), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N = 52 for the APOEε4 non-carriers and N = 23 for the APOEε4 carriers. ApoE4 positive indicates APOEε4 carriers, while ApoE4 negative indicates APOEε4 non-carriers. (a) bar plot shows the difference in Age at Death between APOEε4 carriers and non-carriers. (b) bar plot shows the difference in Age of Onset Cognitive Symptoms between APOEε4 carriers and non-carriers. (c) bar plot shows the difference in Age of Dementia Diagnosis between APOEε4 carriers and non-carriers. (d) bar plot shows the difference in the Last MMSE Score between APOEε4 carriers and non-carriers. (e) bar plot shows the difference in the Interval from the last MMSE in months between APOEε4 carriers and non-carriers.

Table 1. Descriptive statistics of participant characteristics.

		Mean (SD)	Range
Age at death		88.56 (7.89)	35
Sex	Female	46 (61.3%)	\
	Male	29 (38.7%)	\
APOEε4	Carriers	23 (31%)	\
	Non-carriers	52 (69%)	\
Cognitive Status	Dementia	42 (56%)	\
	No Dementia	33 (44.0%)	\
Age of Onset Cognitive Symptoms		81.00 (11.42)	41
CASI Score		86.80 (9.60)	33
MMSE Score		24.42 (4.58)	24
MOCA Score		18.21 (6.47)	21
Year of Education		16.29 (2.78)	9

Note. CASI Score: The Cognitive Abilities Screening Instrument. MMSE Score: Mini-Mental State Examination. MOCA Score: Montreal Cognitive Assessment.

Table 2. The effect size of the impact of the APOEε4 on cognitive function.

	APOEε4 carriers		APOEε4 non-carriers
	M	SD	M	SD	df	t	Cohen’s d
Age at Death	85.43	7.98	89.97	7.52	38.64	2.34	0.58
Age of Onset Cognitive Symptoms	71.92	11.17	85.19	8.96	17.81	3.61	1.37
Age of Dementia Diagnosis	76.70	12.42	86.42	8.27	12.21	2.29	1.02
Last MMSE Score	49.65	6.23	25.53	3.34	24.08	2.34	0.66
Interval from Last MMSE in Months	22.19	32.14	32	25.88	28.94	-2.29	0.79

4.3. The Impact of APOEε4 on AD Patient’s Pathology

Several t-tests were conducted to examine whether there was a significant difference between APOEε4 carriers and non-carriers in participants’ number of AT8 positive cells (tau), 6e10 positive cells (Aβ), 1ba1 and 6e10 positive cells (Aβ), hematoxylin positive nuclei, and glial fibrillary acidic protein (GFAP). The significant difference between APOEε4 carriers and non-carriers that have significant P values (<0.05) and effect sizes (Cohen’s d) is shown in Figure 2 and Table 3. The significant difference in these hallmarks of AD pathology in specific cerebral cortex layers 1 - 5 between APOEε4 carriers and non-carriers that have significant P values (<0.05) is shown in Figure 3.

Figure 2. The P-value of the Impact of APOEε4 on AD Pathology in Grey Matter. Note. From a-e, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N = 52 for the APOEε4 non-carriers and N = 23 for the APOEε4 carriers. ApoE4 positive indicates APOEε4 carriers, while ApoE4 negative indicates APOEε4 non-carriers. (a) The bar plot shows the difference in the number of AT8 positive cells in the grey matter of APOEε4 carriers and non-carriers. (b) bar plot shows the difference in the number of 6e10 positive objects grey matter between APOEε4 carriers and non-carriers. (c) bar plot shows the difference in the number of Iba1 and 6e10 positive co-localized objects grey matter between APOEε4 carriers and non-carriers. (d) bar plot shows the difference in the number of hematoxylin-positive nuclei grey matter between APOEε4 carriers and non-carriers. (e) bar plot shows the difference in the total of glial fibrillary acidic protein (GFAP) grey matter between APOEε4 carriers and non-carriers.

Table 3. The effect size of the impact of APOEε4 on AD pathology in grey matter.

	APOEε4 carriers		APOEε4	non-carriers
	M	SD	M	SD	df	t	Cohen’s d
Number of AT8 Positive Cells Grey Matter	724.21	597.33	185.89	335.21	26.94	−4.17	1.32
Number of 6e10 Grey Matter	13822.30	9901.74	5091.69	9113.34	35.27	−3.73	0.97
Percent of 1ba1 and 6e10 Grey Matter	20.72	12.26	7.68	9.49	32.05	−4.62	1.28
Number of Hematoxylin Grey Matter	102729.96	43364.31	80940.33	29973.8	29.36	−2.24	0.66
Total GFAP Positive Area Grey Matter	7955801	6064636.58	4530796	3966739.33	28.74	−2.53	0.76

Figure 3. The Impact of APOEε4 on AD Pathology in Cerebral Cortex Layers 1-5. Note. From (a)-(g), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N = 52 for the APOEε4 non-carriers and N = 23 for APOEε4 carriers. ApoE4 positive indicates APOEε4 carriers, while ApoE4 negative indicates APOEε4 non-carriers. (a) bar plot shows the difference in the average 6e10 positive objects in cerebral cortex layers 1 - 5 between APOEε4 carriers and non-carriers. (b) bar plot shows the difference in the percent of 6e10 positive co-localized objects grey matter in cerebral cortex layers 1 - 5 between APOEε4 carriers and non-carriers. (c) bar plot shows the difference in the number of Iba1 and 6e10 positive co-localized objects per area grey matter in cerebral cortex layers 1 - 5 between APOEε4 carriers and non-carriers. (d) bar plot shows the difference in the number of Hematoxylin positive nuclei grey matter in cerebral cortex layers 1 - 5 between APOEε4 carriers and non-carriers. (e) bar plot shows the difference in total GFAP positive area grey matter in cerebral cortex layers 1-5 between APOEε4 carriers and non-carriers.

4.4. The Impact of APOEε4 on the Proportion of Neurons in Prefrontal Cortex

Several t-tests were conducted to examine whether there was a significant difference in the proportion of neurons between APOEε4 carriers and non-carriers, including L2/3 IT (Layer 2/3 intra-telencephalic), L5 IT, L4 IT, Sst (somatostatin), L6b, L6 CT (corticothalamic), Lamp5Lhx6, OPC (oligodendrocyte precursor cells), Pvalb (parvalbumin), Vip (vasoactive intestinal polypeptide), L6 IT Car3, L6 IT, Sncg (synuclein gamma), MicrogliaPVM (perivascular macrophages), Pax6, L5 ET (extra-telencephalic projecting), Chandeller, Sst Chodl, VLMC (vascular and leptomeningeal cells), Endothelial, Oligodendrocyte, L5/6 NP (near-projecting), lamp5, and Astrocyte. APOEε4-carriers have a lower proportion of L6b, Sst Chodl, and L5/6 NP than APOEε4 non-carriers, with significant P values (<0.05), and effect size (Cohen’s) as shown in Figure 4 and Table 4.

4.5. The Impact of APOEε4 on the Gene Expression of Degenerating Neurons

The single-cell RNA sequence was conducted to examine whether there was a significant difference in gene expression related to ion channels in Sst chodl, L6 b, and L5/6 NP cells between APOEε4-carriers and non-carriers. In Sst chodl, GLRA1 gene controlling Glycine receptors was found to have significantly less gene expression in APOEε4-carriers than in APOEε4 non-carriers, with P = 0.04 < 0.05. In L6 b, there was no gene expression difference related to ion channels between APOEε4 carriers and non-carriers. In L5/6 NP cells, KCNA1 gene controlling potassium voltage ion channels was found to have significantly less gene expression in the APOEε4 carriers than in the APOEε4 non-carriers, as shown in Figure 5 and Figure 6.

Figure 4. The P-value of the Impact of the APOEε4 on the Proportion of Neurons. Note. From (a)-(f), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N = 52 for the APOEε4-non carriers and N = 23 for the APOEε4-carriers. ApoE4 positive indicates APOEε4 carriers, while ApoE4 negative indicates APOEε4 non-carriers. (a) bar plot shows the difference proportion of L2/3 IT, L5 IT, L4 IT, and Sst cells between APOEε4-carriers and non-carriers. (b) bar plot shows the difference proportion of L6b, L6 CT, Lamp5Lhx6, and OPC cells between APOEε4-carriers and non-carriers. (c) bar plot shows the difference proportion of Pvalb, Vip, L6 IT Car3, and L6 IT cells between APOEε4-carriers and non-carriers. (d) bar plot shows the difference proportion of Sncg, MicrogliaPVM, Pax6, and L5 ET cells between APOEε4-carriers and non-carriers. (e) bar plot shows the difference proportion of Chandeller, Sst Chodl, VLMC and Endothelial cells between APOEε4-carriers and non-carriers. f. bar plot shows the difference proportion of Oligodendrocyte, L5/6 NP, lamp5, and Astrocyte cells between APOEε4-carriers and non-carriers.

Table 4. The effect size of the impact of the APOEε4 on the proportion of neurons.

Neuron	APOEε4 carriers		APOEε4 non-carriers
	M	SD	M	SD	df	t	Cohen’s d
L6 b	0.93	0.53	1.31	0.74	50.25	2.49	0.54
Sst Chodl	0.09	0.03	0.16	0.12	70.41	4.13	0.70
L5/6 NP	1.17	0.38	1.39	0.52	48.49	2.04	0.45

Figure 5. The Difference in GLRA1 Gene Expression between APOEε4 Carriers and Non-carriers. Note. ApoE4 positive indicates APOEε4 carriers, while ApoE4 negative indicates APOEε4 non-carriers.

Figure 6. The Difference in KCNA1 Gene Expression between APOEε4 Carriers and Non-carriers. Note. ApoE4 positive indicates APOEε4 carriers, while ApoE4 negative indicates APOEε4 non-carriers.

5. Discussion

This study investigates the effect of APOEε4 on AD patients’ cognitive function, AD pathology, the number of neurons in DLPFC, and gene expression of ion channels. Our findings suggest that APOEε4 is a factor leading to lower cognitive function and an increased risk of AD pathology. Specifically, APOEε4 is associated with a decline in Sst Chodl, L6b, and L5/6 NP neurons. In Sst Chodl neurons, APOEε4 reduce the expression of the glycine receptors gene, while in L5/6 NP neurons, APOEε4 reduce the expression of the potassium voltage ion channels (Kv1 channels) gene.

The present study highlights the relationship between the expression of APOEε4 and cognitive impairment, as well as the level of markers of AD pathology. The expression of APOEε4 is related to the increasing number of AT8 cells, 6e10 positive cells, 1ba1, 6e10 co-localized cells, and Glial fibrillary acidic protein (GFAP) in the prefrontal cortex. The findings align with previous studies [17]-[20] [27], indicating that APOEε4 carriers have a higher level of Aβ and tau. Additionally, the findings that APOEε4 carriers have a younger age at death, lower ability of cognitive function, and a younger age of onset of dementia are consistent with previous studies [14]-[16]. Overall, these findings validate the idea that APOEε4 increases the risk of AD from accumulation of Aβ and tau and reduces people’s cognitive function.

This study shows a link between APOEε4 and neurodegeneration. Focusing on the cell-type-specific evidence, APOEε4 leads to damage in Sst Chodl, L5/6 near-projecting pyramidal neurons (L5/6 NP), and L6b neurons. These findings are consistent with previous studies that AD is accompanied by neurodegeneration of these neurons [37] [38]. Sst neurons regulate the degradation of Aβ [42], and the loss of Sst neurons in AD patients has been shown to be related to the accumulation of Aβ, which increases the risk of AD [37]. Sst Chodl is a subtype of Sst neuron, which is an inhibitory neuron [43] [44]. Similarly to Sst neurons, Sst Chodl neurons also degenerate in AD patients [38], and in this study, APOEε4 carriers are more likely to accumulate Aβ than APOEε4 non-carriers, indicating that Sst Chodl neurons may also have a similar function as Sst neurons to clearance of Aβ and APOEε4 lead to the degenerate of Sst Chodl neurons, resulting the accumulation of Aβ contributing to AD’s cognitive impairment [37] [38]. L5/6 NP and L6b are neuronal glutamatergic neurons and thus play a role in releasing glutamate [45]. In this study, APOEε4 leads to damage in L5/6 NP and L6b and the accumulation of Aβ, leading to glutamatergic dysfunction, contributing to synapse loss, synapse toxicity, and cognitive impairment related to AD [34]-[36] [46]-[48].

This study uses single-cell RNA sequencing to explore how APOEε4 affects the gene expression in specific neuron types, including Sst Chodl and L5/6 near-projecting pyramidal neurons (L5/6 NP). In Sst Chodl neurons, APOEε4 was associated with reduced expression of the glycine receptors. Glycine receptors are crucial for neuronal inhibition, as their activation allows Cl^- ions to influx into the postsynaptic membrane, hyperpolarizing it and reducing neuronal excitability [49] [50]. A decrease in glycine receptors weakens neuronal inhibition. Additionally, intracellular Aβ has been linked to inhibition loss regulated by the glycine receptors [30]. Our study showed that APOEε4 promotes Aβ deposition, which in turn decreases gene expression of glycine receptors in the Sst Chodl neurons, leading to reduced inhibition and increased neuronal excitability. This results in the imbalance between inhibition and excitability [30] [51], contributing to cognitive impairments related to AD [47] [48]. In addition, we found that in L5/6 NP neurons, APOEε4 carriers decrease the expression of potassium voltage ion channels (Kv1 channels). These channels play a critical role in regulating the action potential threshold, neuron excitability, and synaptic transmission [52]-[54]. Reduced Kv1 channel activity could increase the probability of neurotransmitter release [31]-[33]. The current study showed APOEε4 decreases gene expression of Kv1 channels, which may prompt L5/6 NP neurons to release glutamate. Additionally, APOEε4 carriers lead to Aβ accumulation [17]-[20], interrupting the glutamate uptake and clearance [55] [56]. Together, APOEε4 decreases gene expression of Kv1 channels and prompts the accumulation of Aβ, which could elevate extracellular glutamate levels, resulting in excitotoxicity, synapse toxicity and synapse loss [34]-[36]. These downstream may lead to cognitive impairments observed in AD, consistent with previous studies [46]-[48] [57].

6. Conclusion

Our study demonstrates that APOEε4 contributes to cognitive impairment, shorter life span, and earlier onset of AD. The APOEε4 also leads to neurodegeneration related to AD in Sst cells, L5/6 NP, and L6b neurons. The APOEε4 decreases the gene expression of glycine receptors in Sst cells and the gene expression of potassium voltage ion channels in L5/6 NP, which is related to cognitive impairment in AD. The findings provided insight into how APOEε4 contributes to AD symptoms, neuronal loss, and altered gene expression. Our study strengthens the understanding of the cause of AD from a gene perspective and offers a new clue for the future intervention of AD, such as through enhancing and recovery of the function of ion channels to alleviate AD patients’ cognitive impairment. However, this study only focused on the role of the APOEε4 in the prefrontal cortex. Different brain regions have different functions and have various amounts of different neurons [58]. Therefore, APOEε4 may have a different effect on the number of neurons between various brain regions. The damaged temporal cortex and parietal cortex are related to the early stage of AD [59] [60]. Hence, future studies should focus on the impact of APOEε4 on the temporal and parietal cortex neurons, particularly in the early stage of AD. Additionally, one limitation is the smaller sample size of APOEε4 carriers in the SEA-AD dataset, and thus, future studies should include more APOEε4 carriers to gain a more general understanding of the impact of APOEε4 on AD patients’ cognitive impairment.

Conflicts of Interest

The author declares no conflicts of interest regarding the publication of this paper.

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