Millions of individuals carry the APOE4 gene variant, recognized as the most significant genetic predisposition to Alzheimer’s disease, and new scientific inquiry suggests this genetic factor may instigate alterations in brain function years prior to the emergence of discernible memory impairments. Researchers at the Gladstone Institutes have meticulously charted a molecular pathway that potentially elucidates these nascent effects, with their discoveries also indicating a promising avenue for rectifying some of these observed changes.
The investigation, conducted using preclinical models and detailed in a recent publication in Nature Aging, revealed that the APOE4 variant prompts an elevated synthesis of a specific protein known as Nell2. Consequently, increased concentrations of Nell2 were found to induce a reduction in the physical size of neurons and an abnormal state of heightened electrical activity. Strikingly, the study observed a correlation wherein mice exhibiting more pronounced brain hyperactivity during their early developmental stages subsequently displayed more severe memory deficits in later life.
A pivotal aspect of this research involved the deliberate reduction of Nell2 production. The findings demonstrated that even in adult mice predisposed by APOE4, their neurons reverted towards a more typical size and exhibited normalized firing patterns. This outcome offers a compelling rationale for the potential development of therapeutic interventions targeting Nell2, which could subsequently aid individuals carrying the APOE4 variant in mitigating their heightened susceptibility to Alzheimer’s disease.
"To our knowledge, this marks the inaugural study to directly investigate the functional impact of APOE4 on neuronal activity across various age stages," stated Misha Zilberter, PhD, a principal staff research scientist at Gladstone and a senior author of the research. "We uncovered fundamental modifications within neural circuits in young mice that still exhibited unimpaired learning and memory capabilities. Crucially, these alterations were found to be predictive of subsequent cognitive impairments observed in older age."
APOE4 represents one of the three prevalent isoforms of the APOE gene, yet it exerts a substantially more pronounced influence on Alzheimer’s risk compared to its counterparts. An estimated quarter of the global population carries the APOE4 variant, and it is present in a significant majority, ranging from 60% to 75%, of individuals diagnosed with Alzheimer’s disease.
"This research represents a significant advancement for the field of Alzheimer’s disease investigation," commented Yadong Huang, MD, PhD, associate director of the Gladstone Institute of Neurological Disease and a senior author of the study. "It paves the way for a more profound understanding of how APOE4 alters neuronal function during younger years, thereby increasing the risk of cognitive deterioration. Furthermore, it opens avenues for the development of therapeutic strategies capable of counteracting the detrimental effects of APOE4 at an early stage."
Prior scientific endeavors had already indicated the presence of unusually elevated brain activity in human carriers of APOE4 preceding middle age, with this early hyperactivity also being linked to subsequent cognitive decline. However, the precise mechanisms by which APOE4 precipitates these cellular changes and their contribution to later memory issues remained elusive.
To address these questions, the research team meticulously analyzed electrophysiological recordings of brain activity in young mice and conducted detailed examinations of individual neurons. Their findings indicated that young mice possessing the APOE4 variant exhibited excessive neuronal firing within two specific regions of the hippocampus, a brain area critically involved in memory formation and retrieval.
Significantly, these same hippocampal regions have been previously identified as exhibiting hyperactivity in human APOE4 carriers. "We observed that the degree of hyperactivity in young mice was directly correlated with their performance on spatial learning and memory tasks later in life," explained Dennis Tabuena, PhD, a scientist jointly mentored by Zilberter and Huang, and the lead author of the new publication.
For comparative analysis, the researchers contrasted these animals with mice carrying the APOE3 variant, a form of the APOE gene generally associated with a lower incidence of Alzheimer’s disease in humans. The neurons within the affected hippocampal areas were found to be smaller in APOE4 mice when compared to their APOE3 counterparts. Smaller neuronal structures are known to be more responsive to stimulation, thereby increasing their propensity for overactivity. In contrast, hippocampal neurons in APOE3 mice did eventually display increased excitability, but this phenomenon did not manifest until the animals reached an advanced age.
"This suggests that APOE4 accelerates a process that mimics normal aging, potentially explaining why individuals with this gene variant are more predisposed to developing Alzheimer’s disease at an earlier stage of life," Dr. Huang elaborated.
A crucial insight from the study pertains to the origin of these APOE4-induced effects. While the majority of APOE4 in a healthy brain is produced by astrocytes, supportive cells for neurons, researchers had long hypothesized that astrocytes were primarily responsible for the association between APOE4 and Alzheimer’s risk. However, the current findings suggest a different locus of action. The research indicates that the hippocampal hyperactivity linked to APOE4 is predominantly driven by APOE4 produced intrinsically within the neurons themselves.
"When we genetically removed the APOE4 gene from astrocytes, there was no discernible change in neuronal function," Dr. Zilberter reported. "However, upon its deletion from neurons, these cells resumed their normal size and exhibited normalized functionality."
The researchers then delved into identifying the specific molecular cascade responsible for the reduction in size and increased excitability of APOE4-bearing neurons. They meticulously analyzed patterns of gene expression within individual cells across various hippocampal cell types. This detailed analysis pinpointed Nell2 as a key player, with abnormally elevated levels detected in neurons carrying the APOE4 variant.
Subsequently, the research team employed CRISPRi, a gene-editing technique that can effectively suppress gene activity without permanently altering the DNA sequence, to reduce Nell2 levels in hippocampal neurons derived from adult APOE4 mice. Following the reduction of Nell2, the neurons demonstrated a return towards their normal size and exhibited diminished excitability. This critical finding strongly supports the hypothesis that elevated Nell2 is the direct cause of the excessive neuronal activity observed in brains harboring the APOE4 variant.
While Nell2 had not been previously investigated in direct relation to APOE4, earlier studies had reported increased concentrations of this protein in the brains of individuals with Alzheimer’s disease, with higher levels correlating with poorer cognitive function. "The most encouraging aspect of our findings regarding Nell2 is our ability to reverse the disease-related manifestations in adult mice by lowering its levels," Dr. Huang emphasized. "This indicates that the observed damage is not necessarily irreversible, and there may indeed be a therapeutic window for intervention even after disease processes have been initiated."
The research was made possible through funding from several prestigious national institutions, including the National Institute on Aging (grant numbers R01AG061150, R01AG087323, R01AG092390, R01AG085468, R01AG055682, R01AG071697, P01AG073082, F32AG0859612), the National Institute of Neurological Disorders and Stroke (grant number K99NS134734), and the National Center for Research Resources (grant number C06 RR018928).
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