Re: 중추신경(선조체, 편도체, 후각피질, 시상하부) 뇌신경이 재생된다 2024년 NATURE

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Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease

The hippocampus is one of the most affected areas in Alzheimer’s disease (AD)1 . Moreover, this structure hosts one of the most unique phenomena of the adult mammalian brain, namely, the addition of new neurons throughout life2 . This process, called adult hippocampal neurogenesis (AHN), confers an unparalleled degree of plasticity to the entire hippocampal circuitry3,4. Nonetheless, direct evidence of AHN in humans has remained elusive. Thus, determining whether new neurons are continuously incorporated into the human dentate gyrus (DG) during physiological and pathological aging is a crucial question with outstanding therapeutic potential. By combining human brain samples obtained under tightly controlled conditions and state-of-the-art tissue processing methods, we identified thousands of immature neurons in the DG of neurologically healthy human subjects up to the ninth decade of life. These neurons exhibited variable degrees of maturation along differentiation stages of AHN. In sharp contrast, the number and maturation of these neurons progressively declined as AD advanced. These results demonstrate the persistence of AHN during both physiological and pathological aging in humans and provide evidence for impaired neurogenesis as a potentially relevant mechanism underlying memory deficits in AD that might be amenable to novel therapeutic strategies.

해마는 

알츠하이머병(AD)에서 가장 영향을 많이 받는 부위 중 하나입니다1 . 

또한 이 구조는 성인 포유류의 뇌에서 

가장 독특한 현상 중 하나인 일생 동안 

새로운 뉴런이 추가되는 현상을 주관합니다2 . 

성인 해마 신경 발생(AHN)이라고 불리는 이 과정은 

전체 해마 회로에 비할 데 없는 수준의 가소성을 부여합니다3,4. 

그럼에도 불구하고 

인간의 AHN에 대한 직접적인 증거는 아직까지 밝혀지지 않았습니다. 

따라서 

생리적 및 병리적 노화 과정에서 새로운 뉴런이 

인간의 치상회(DG)에 지속적으로 통합되는지를 확인하는 것은 

뛰어난 치료 잠재력을 가진 중요한 문제입니다. 

저희는 엄격하게 통제된 조건에서 얻은 

인간의 뇌 샘플과 최첨단 조직 처리 방법을 결합하여 

신경학적으로 건강한 사람의 생후 9년까지의 

DG에서 수천 개의 미성숙 뉴런을 확인했습니다. 

이 뉴런들은 

AHN의 분화 단계에 따라 다양한 수준의 성숙도를 보였습니다. 

대조적으로, 

알츠하이머병이 진행됨에 따라 

이러한 뉴런의 수와 성숙도는 점차 감소했습니다.

 이러한 결과는 

인간의 생리적 및 병리적 노화 동안 AHN의 지속성을 입증하고 

새로운 치료 전략에 적용될 수 있는 

AD의 기억력 결손과 관련된 잠재적 메커니즘으로서 

신경 발생 장애에 대한 증거를 제공합니다.

The occurrence of AHN in humans was first shown by Eriksson et al.5 . Their findings have been supported6–8 and questioned9,10 by a number of subsequent studies. However, the limited availability of adequately preserved human brain tissue samples, together with the heterogeneity of tissue processing methodologies, is considered to have contributed to a lack of consensus in this regard11. To shed light on the potential occurrence of AHN during physiological human aging, we sought to determine whether the adult human DG harbors a population of immature neurons throughout life (Fig. 1). To this end, we first established the most suitable conditions in which to study AHN in humans. We used brain samples obtained under tightly controlled conditions and state-of-the-art tissue processing methodologies (Methods and Extended Data Figs. 1–6). These conditions involved careful eval‎uation of medical records, exclusion of subjects showing any neurological disease or cognitive disability, and confirmation of Braak stage 0 by neuropathological examination (Methods). We also monitored the post-mortem delay (PMD; the time lapse between exitus and tissue immersion in fixative), optimized fixation time and conditions, and avoided freezing, paraffin inclusion, or any type of mechanical alteration of the tissue. Under these methodological constraints, we identified thousands of doublecortin-expressing (DCX+) neurons in the DG for a cohort of 13 neurologically healthy subjects between 43 and 87 years of age (Fig. 1d,h,j and Extended Data Figs. 1–4 and 6c). These findings were further confirmed using four anti-DCX antibodies raised against different parts of the protein (Extended Data Fig. 4). We noted that the DCX signal was absent in non-neurogenic brain regions such as the entorhinal cortex (EC) (Fig. 1b,f and Extended Data Fig. 6a) and in the CA1 (Fig. 1c,g and Extended Data Fig. 6b) and CA3 (Fig. 1e,i and Extended Data Fig. 6d) hippocampal subfields. AHN is a tightly regulated process that encompasses a wellcharacterized sequence of maturation stages in rodents12. After exiting the cell cycle, immature neuroblasts go through a series of differentiation phases before becoming fully mature. In rodents, DCX expression‎ occurs during most of the differentiation stages of AHN, and the population of DCX+ cells in consequence exhibits varying degrees of maturation11–13. Therefore, we addressed whether cell subpopulations at distinct stages of maturity could also be distinguished among DCX+ cells in the human DG (Fig. 2). To this end, we first analyzed the expression‎ of cell markers characteristic of the different maturation stages of AHN11,12 in human DCX+ cells (Fig. 2a–i). Notably, 91% of DCX+ cells also expressed prospero homeobox 1 (Prox1) (Fig. 2c,i), thereby suggesting that most DCX+ cells had already acquired a dentate granule cell (DGC) fate14 and validating DCX as a reliable marker of immature DGCs in humans. Accordingly, a subset of DCX+ cells were positive for markers transiently expressed by immature neurons, such as polysialic acid–neural cell adhesion molecule (PSA-NCAM) (Fig. 2d,i) and calretinin (CR) (Fig. 2e,i). Approximately 40–60% of DCX+ cells expressed markers of neuronal identity such as neuronal nuclei (NeuN) (Fig. 2f,i), βIII-tubulin (Fig. 2g,i), and tau (Fig. 2i). Finally, ~40% of DCX+ cells expressed markers of more differentiated neurons, such as calbindin (CB) (Fig. 2h,i). During AHN in rodents, differential expression‎ of two calciumbinding proteins, CR and CB, defines two postmitotic maturation stages of DGCs12,15. CR is transiently expressed by immature neurons during early differentiation phases, whereas CB expression‎ is characteristic of more differentiated DGCs15. Expression‎ of these two cell markers also correlates with different morphological features16. Accordingly, remarkable morphological heterogeneity was observed among DCX+ cells in the human DG (Fig. 2k–m). Thus, we questioned whether this morphological heterogeneity also reflects distinct maturation stages. To examine this, we analyzed the morphological features of double-labeled (DCX+CR+ and DCX+CB+) cells. Notably, DCX+CR+ cells were predominantly located at the hilar border of the granule cell layer (GCL) (Fig. 2k,o), referred to as the subgranular zone (SGZ). They had smaller soma (Fig. 2k,n), an elongated morphology characteristic of immature DGCs (Fig. 2k), and ~2 primary neurites per cell (Fig. 2k,p), which were mainly oriented parallel to the SGZ (Fig. 2k,q). These observations support the notion that DCX+CR+ cells have an immature phenotype16. In contrast, most DCX+CB+ cells occupied deeper positions within the GCL (Fig. 2l,o). They also exhibited larger cell soma (Fig. 2l,n), an oval-to-round morphology (Fig. 2l), and one primary apical neurite (Fig. 2l,p), which was mainly oriented perpendicular to the SGZ, toward the molecular layer (ML) (Fig. 2l,q). These features correlate with a more mature phenotype for this cell population16. Altogether, these data strongly support the notion that subpopulations of DCX+ cells have a variable degree of maturation in the human DG. Although birthdating analyses are required to determine the exact duration of individual maturation stages for human DGCs, completion of the maturation process has been proposed to take several months17 or years9 in primates. The relative abundance of DCX+ immature neurons detected, together with expression‎ of cell markers characteristic of both early and late stages of maturation, suggests that these cells also have an extended maturation period during AHN in humans. On the basis of both the percentages of double-labeled cells and their morphological features, here we outline the first model of the differentiation stages of human AHN (Fig. 2j). Our data support the persistence of AHN in the adult human DG until the ninth decade of life. However, recent studies failed to detect high numbers of DCX+ cells in the human hippocampus9,10. Therefore, we examined the extent to which heterogeneity in tissue processing could account for the different results. We found that detection of AHN markers in the human DG is critically dependent on fixation conditions and histological pretreatment of the tissue (Extended Data Figs. 2–5 and 7). Our data demonstrate that the prolonged or uncontrolled fixation conditions to which human samples are typically exposed in brain banks worldwide lead to a sharp reduction in the number of DCX+ cells detected in the adult DG (Extended Data Fig. 2). Nonetheless, implementation of the most suitable tissue processing methodologies unraveled the presence of thousands of DCX+ cells with an unambiguously identifiable neuronal morphology in the adult DG. Given this observation, AHN clearly emerges as a robust phenomenon during physiological aging in humans. The hippocampus is one of the brain regions most affected in AD1,18, and DGCs exhibit remarkable morphological alterations in patients with AD18. Given the role that immature neurons have in hippocampus-dependent learning3 , as well as the alterations in AHN observed in numerous animal models of the disease19, we decided to study this process in a cohort of 45 patients with AD between 52 and 97 years of age distributed among the six neuropathological Braak stages of the disease20 (Fig. 3a–c and Extended Data Figs. 7a,b and 8a–g). Stereological estimation of the number of DCX+ cells revealed a marked and progressive decline in this number as the disease advanced (Fig. 3d–k). We also observed that the number of DCX+ cells decreased moderately as age increased from 40 to 90 years in neurologically healthy control individuals (Extended Data Fig. 1b). These data are in agreement with previous evidence suggesting a modest decline in the rate of AHN during physiological aging in humans6 . However, the number of DCX+ cells detected in neurologically healthy individuals of any age was consistently higher than that found in patients with AD, regardless of the age of these patients (Fig. 3l). These data strongly support the notion that AD is a condition that differs from physiological aging and suggest that, despite a physiological age-related decline in the population of DCX+ cells, independent neuropathological mechanisms contribute to devastating the population of immature neurons in AD. Given that newborn neurons that do not adequately complete maturation and synaptic integration programs are promptly eliminated during AHN in rodents12,21, a reduction in either the addition or the survival of immature neurons might underlie the marked decline in cellular counts observed in patients with AD. Thus, we considered it pertinent to determine whether a blockade occurs in the maturation of DCX+ cells in the DG of patients with AD. To this end, we studied the expression‎ of cell markers related to distinct maturation stages during AHN8,11,12 in DCX+ cells from these patients (Fig. 4). We detected a reduction in the percentage of DCX+ cells that expressed PSA-NCAM (Fig. 4a,d) starting at Braak stage III. This was followed by a reduction in the expression‎ of Prox1 (Fig. 4f), NeuN (Fig. 4g), βIII-tubulin (Fig. 4h), and CB (Fig. 4b,i) at some of the subsequent stages of the disease. These data provide evidence of substantial impairment of the maturation of DCX+ cells as AD advances. Given the unique electrophysiological properties of immature DGCs in rodents22,23, the aforementioned alterations could be related to the selective impairment of certain types of hippocampus-dependent learning observed in patients with AD24. Notably, alterations in AHN were detected at early stages of the disease, even before the generalized presence of neurofibrillary tangles or senile plaques in the DG (Fig. 3a–c and Extended Data Fig. 8). Similarly, various alterations have been proposed to occur during the prodromal stages of the disease, which are believed to start several decades before manifestation of the first clinical symptoms25. Therefore, early detection of AHN impairments by noninvasive methods might allow these alterations to be used as relevant biomarkers of the advance of the disease. Moreover, therapeutic strategies aimed at increasing the numbers and functionality of these cells might be relevant to prevent or slow down AD progression. Our data bring to light the existence of a dynamic population of immature neurons in the human DG throughout physiological and pathological aging until the tenth decade of life. This finding points to unexplored mechanisms of circuit plasticity in the aging human hippocampus. Of note, our results demonstrate a profound, multifaceted impairment of AHN in patients with AD starting at early stages of the condition. Therefore, restoration of normal levels of AHN in these patients emerges as a potential therapeutic approach to counteract the progression of this as yet incurable disease. Online content Any methods, additional references, Nature Research reporting summaries, source data, statements of data availability and associated accession codes are available at https://doi.org/10.1038/ s41591-019-0375-9

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