Dendritic spine degeneration: a primary mechanism in the aging process

Abstract

Recent reports suggest that aging is not solely a physiological process in living beings; instead, it should be considered a pathological process or disease (Amorim et al., 2022). Consequently, this process involves a wide range of factors, spanning from genetic to environmental factors, and even includes the gut microbiome (GM) (Mayer et al., 2022). All these processes coincide at some point in the inflammatory process, oxidative stress, and apoptosis, at different degrees in various organs and systems that constitute a living organism (Mayer et al., 2022; Aguilar-Hernández et al., 2023). However, one of the most studied organs in the aging process is the brain, due to the cognitive deficits observed in aging animals, including humans (Aguilar-Hernández et al., 2023). Moreover, with aging, a set of both metabolic and cardiovascular diseases manifests, among which are diabetes mellitus and high blood pressure. With the progression of both aging and these diseases, cognitive deficits have been demonstrated in both human and animal models (Flores-Gómez et al., 2019; Flores et al., 2020). These cognitive deficits can vary depending on the degree of affectation to interneuronal communication, specifically at the level of dendritic spines (Aguilar-Hernández et al., 2023). A recent study conducted by our research team has revealed that aging results in a decline in dendritic spine density and a reconfiguration of dendritic spine shapes in corticolimbic areas in rodents, such as the prefrontal cortex (PFC). The PFC plays a crucial role in cognitive functions like attention, decision-making, and control over reward and motivation (Reyes-Lizaola et al., 2024). While there is extensive data on the decrease in dendritic spine density due to aging in humans (Aguilar-Hernández et al., 2023), the impact of aging on the structural neuroplasticity of dendritic spines remains unexplored, to the best of our knowledge. To address this matter, postmortem human brain samples were subjected to the Golgi-Cox technique to assess the structural neuroplasticity of pyramidal cells of the PFC, specifically of the orbitofrontal cortex (OFC) region, during aging. For this investigation, ten samples from the OFC were obtained postmortem, divided into two groups: young adults (n = 5, mean age ± standard deviation = 20.67 ± 1.085 years; male:female ratio = 4:1) and adults (n = 5, mean age ± standard deviation = 53.83 ± 2.33 years; male:female ratio = 3:2). These OFC samples, approximately 16 cm³ in size and containing both white and gray matter, were collected through autopsies at the “Instituto de Ciencias Forenses” in Mexico City, under the supervision of a certified coroner. The selected samples represented various causes of death, including traffic accidents, heart attacks, and carbon monoxide asphyxia, with no recorded history of substance abuse or diagnosed psychiatric disorders. The OFC samples then underwent a modified Golgi-Cox procedure, adapted from the method designed for the entire rat brain (Tendilla-Beltrán et al., 2019; Aguilar-Hernández et al., 2020). In brief, the samples were immersed in the Golgi-Cox solution for 30–50 days. Following this, they were transferred to a 30% sucrose solution for one week to facilitate sectioning. Slices with a thickness of 250 μm were obtained using a manual vibratome (Campden Instruments) and were placed on gelatin-coated slides. Subsequently, these slices underwent a reduction of the Golgi-Cox solution with ammonium hydroxide for 30 minutes, followed by treatment with Kodak rapid fixative for an additional 30 minutes. Finally, the samples were dehydrated using progressively concentrated ethanol solutions and ultimately mounted with synthetic resin. The analysis of dendritic spines was conducted by identifying pyramidal neurons from layers 3 (L3) and 5 (L5) of the OFC using an optical microscope equipped with a camera lucida (Leica Microsystems). The focus of the analysis was on the number and morphological classification of dendritic spines located in the distal segments of the basilar arbor, as previously reported (Tendilla-Beltrán et al., 2019). To quantify the dendritic spine density per 10 μm, the number of dendritic spines in the 30 μm most distal dendrite and the average of the three 10 μm-length segments were reported. Within the same dendrite, it was categorized a hundred consecutive dendritic spines based on their morphological characteristics into: mushroom (M), spines with prominent heads with a well-defined neck; thin (T), elongated structures with a similar head/neck diameter; stubby (S), spines lacking a discernible neck; bifurcated (B), spines with a branching neck with distinguishable heads each one; and unclassified (U) for those that did not fit into the aforementioned categories (Figure 1A). The results of spine classification are presented as a relative percentage proportion. Ten neurons per case were analyzed, resulting in a total of 100 neurons included in the study. The analysis of dendritic spines was carried out by a trained observer blinded to experimental groups. Finally, the statistical analysis of the data was performed using either an unpaired t-test or a 2-way ANOVA (considering age and type of spine as independent factors) in GraphPad Prism 9.0 software. The results are expressed as the mean ± the standard error of the mean. All these procedures received approval from the Research Ethics Committee of the “Instituto de Ciencias Forenses del TSJCDMX” (Conbiética-09-CEI-022-20160823). Additionally, this study was conducted in accordance with the National Guide for the Execution of Research Projects of Health in Human Beings (Norma Oficial Mexicana NOM-012-SSA3-2012) and the Helsinki Declaration of 1975.Figure 1: Dendritic spine plasticity in aging.(A, B) Upper panels in A, representative photomicrographs depict pyramidal neurons from layer 3 (L3) of the orbitofrontal cortex (OFC) in both young adults (mean age ± standard deviation = 20.67 ± 1.085 years) and adults (mean age ± standard deviation = 53.83 ± 2.33 years; scale bars: 50 μm). Below panels in A, representative photomicrographs of basilar distal dendritic segments of OFC pyramidal cells from layers 3 and 5 (L5), and a dendritic spine morphological classification scheme indicates mushroom spines (M) in blue, thin spines (T) in green, and stubby spines (S) in red. Additionally, bifurcated (B) and unclassified spines (U) were classified, for details see the main text (scale bar = 10 μm). Unpublished data. (B) Analyses of dendritic spine density and morphological classification. In both OFC layers, the pyramidal cells of the adult group exhibit a smaller number of dendritic spines and an increased proportion of mushroom spines compared with the young adults. Additionally, neurons in OFC L3 show a lower proportion of mushroom spines in the adult group. Unpublished data. (C) Dendritic spine dynamics mechanism. The process of dendritic spine formation initiates with a mobile protuberance known as a filopodium. Upon contact with an axonal button, it establishes a synapse and evolves into a thin dendritic spine. If this thin spine receives consistent stimulation, and neurotrophins, and exists in a healthy environment, it undergoes a long-term potentiation (LTP) process, transforming into a mushroom spine. Under these conditions, the mushroom dendritic spine may further develop into bifurcated spines, ultimately giving rise to two dendritic spines, thereby increasing the overall spine density. Conversely, when a thin dendritic spine receives insufficient axonal stimulation, limited neurotrophins, or exists in an environment characterized by constant oxidative stress and inflammation, it degenerates into a stubby spine through a long-term depression (LTD) process. Stubby spines are non-functional and can contribute to a reduction in dendritic spine density. This last panel of the figure was created with BioRender.com.The findings derived from the assessments of structural neuroplasticity in the distal dendritic spines of pyramidal cells within the OFC reveal a notable reduction in the number of dendritic spines in both OFC L3 and OFC L5 when comparing the adult cohort with the group of young adults (Figure 1B). In the examination of the dendritic spine populations according to their shape (Figure 1B), in OFC L3 (Figure 1B), it is observed that the adult group exhibits a diminished proportion of mushroom spines and an increased presence of stubby spines in comparison to the young adult cohort (Figure 1B). No discernible alterations were noted in the prevalence of other spine types in OFC L3. In the context of OFC L5 (Figure 1B), the adult group demonstrated a decreased proportion of thin spines accompanied by an elevated proportion of stubby spines relative to the young adult group. The other categories of dendritic spines remained unchanged between the two groups. Dendritic spines, or dendritic protrusions, were initially described by Cajal and constitute excitatory sites of the glutamatergic type (Reyes-Lizaola et al., 2024). Various factors regulate the formation and maintenance of these excitatory structures, including neurotrophins, nitric oxide, neurotransmitters such as glutamate, dopamine, and serotonin, as well as the complement system (Flores-Gómez et al., 2019; Aguilar-Hernández et al., 2020; Flores et al., 2020). Notably, neurotrophins, such as nerve growth factor and brain-derived neurotrophic factor, have been extensively studied. Recent reports indicate that brain-derived neurotrophic factor not only participates in the formation of dendritic spines but also plays a crucial role in their maintenance. These data, while preliminary, elucidate that in aging human individuals, there is a discernible augmentation in the number of stubby spines, coupled with a concomitant decline in mushroom or thin spines within postmortem samples of the OFC. These alterations coincide with an overall reduction in the number of dendritic spines (Figure 1B), a phenomenon also documented in other corticolimbic regions (Aguilar-Hernández et al., 2023). Mushroom spines, recognized as mature and functionally synaptic structures, play a crucial role in long-term potentiation, processes integral to memory and learning (Figure 1C). Despite, stubby spines are crucial for spine-dendrite Ca2+ dynamics, which is an essential phenomenon for synaptic plasticity, these structures exhibit poor assembly, and its density increases in the long-term depression processes, which represents a weakness of the synaptic transmission. And for neural ensembles to function a long-term potentiation-long-term depression balance is required, however, stubby spines may be promoting long-term depression and consequently dysfunctional circuits (Falcón-Moya et al., 2020; Figure 1C). Notably, in animal models with implications for schizophrenia, such as neonatal ventral hippocampus lesion, it has been demonstrated that there is a reduction in dendritic spine density, accompanied by an increase in stubby spines and a decrease in mushroom spines, leading to robust cognitive impairments, lighting out the functional implications of the proportion of these dendritic spines in the PFC (Tendilla-Beltrán et al., 2019). In this context, a recent study by Ling et al. (2024) has demonstrated that the PFC of individuals with schizophrenia and older adults shares transcript characteristics related to plasticity that are remarkably similar, not only in neurons but also in astrocytes. This forms a functional circuit between these cells, denominated the synaptic neuron–astrocyte program, which is impaired both in schizophrenia and aging. Another profound instance of cognitive impairment is observed in Alzheimer’s disease. In both human and animal models of Alzheimer’s disease, a significant reduction in the number of neurons has been reported, accompanied by a decrease in dendritic spine density, particularly affecting mushroom spines (Boros et al., 2017). Moreover, synaptic plasticity can be modulated by a myriad of mechanisms, including inflammation and oxidative stress. Recent studies have highlighted the involvement of the immune system in synaptic-related processes, such as synaptic pruning — a process that reduces dendritic spines during puberty, facilitated by the complement system. Additionally, reports suggest interactions among the brain, gut, and gut microbiota, with the immune system playing a role. The GM can produce substances, such as indole molecules, known for their antioxidant and anti-inflammatory properties. As individuals age, the composition of the GM changes, leading to a reduction in the production of these beneficial molecules (Mayer et al., 2022). Consequently, there is an increase in inflammatory and oxidative stress processes, involving nitric oxide synthases. The heightened activity of synthases results in an increase in nitric oxide levels, contributing to a reduction in the number of dendritic spines. It has also been reported that elevated nitric oxide levels may be implicated in the expression of different types of dendritic spines. This is evident in certain aging models and the animal model of neonatal neonatal ventral hippocampus lesion, where an increase in nitric oxide levels has been observed (Morales-Medina et al., 2021). In conclusion, with aging, poorly assembled and non-functional spines, such as stubby-shaped ones, increase, while functional and mature spines, such as mushroom-like ones, decrease – a process that can be referred to as dendritic spine degeneration, and this phenomenon can be modulated by multiple other processes beyond the inherent of the plasticity-related ones (Figure 2).Figure 2: Dendritic spine degeneration in aging.In the aging brain cortical neurons lose not only dendritic spine density, but also thin (T) and mushroom (M) spine proportion decrease. These types of spines represent the formation and strengthening of the synaptic transmission respectively. Additionally, the proportion of stubby spines (S), which represent the weakening of synaptic transmission increases. We coin the dendritic spine degeneration term for referring to all these structural neuroplasticity changes. Moreover, the characteristic processes of aging include increased oxidative stress as a consequence of excessive NO production and sustained inflammation in nervous tissue. Also, there is a continuous release of proinflammatory factors resulting from the infiltration of microbiota and its metabolites due to a disruption of the intestinal epithelium. These cytokines infiltrate the nervous tissue, exacerbating the inflammatory process and oxidative stress. Whether acting individually or in combination, these factors induce alterations in dendritic spine morphology, leading to an increase in stubby spines and a decrease in mushroom spines, ultimately reducing the overall density of dendritic spines. This phenomenon is referred to as dendritic spine degeneration. Created with BioRender.com. NO: Nitric oxide; NOS: nitric oxide synthase.In light of the aforementioned, it is imperative to develop pharmacological tools that augment the formation and maintenance of mushroom spines during the aging process. Notably, drugs possessing anti-inflammatory attributes, such as phenylbutyrate, or exhibiting antioxidative activity, such as resveratrol, have demonstrated the capacity to increase the number of mushroom spines in aged rodents (Flores-Gómez et al., 2019; Flores et al., 2020). This underscores the significance of these processes in neuronal plasticity. Furthermore, it is crucial to enhance the GM to elevate the production of substances with anti-inflammatory and antioxidant properties at the cerebral level. Investigating the GM and the factors contributing to an enhanced inflammatory process at the neuronal level represents an urgent research imperative. Simultaneously, the ongoing development of products endowed with neurotrophic, antioxidant, and anti-inflammatory properties at the neuronal level should be pursued. In conclusion, as pyramidal neurons in the cerebral cortex age, both the density of dendritic spines decreases and the proportion of mushroom spines (associated with synaptic strengthening) declines, while the number of stubby spines (associated with synaptic weakening) increases. We term this phenomenon dendritic spine degeneration. This process, influenced by factors like sustained inflammation or GM impairments typical of aging, leading to oxidative stress, suggests antioxidant molecules could mitigate neuroplasticity changes in aging. GF, FGD, HN, and HTB acknowledge the CONAHCYT’s “Sistema Nacional de Investigadoras e Investigadores” program for membership. AJVH acknowledges CONAHCYT for scholarship. This work was funded by CONAHCYT grant (252808) to GF, CONAHCYT’s “Estancias Posdoctorales por México” program (662350) to HTB. The funding institution did not play anyadditional role in manuscript conception, data collection, analysis, interpretation, manuscript writing, or the choice to submit the work for publication. C-Editors: Zhao M, Liu WJ, Qiu Y; T-Editor: Jia Y

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