In most mammals, neurons are added throughout life in the hippocampus and olfactory bulb. One area where neuroblasts that give rise to adult-born neurons are generated is the lateral ventricle wall of the brain. We show, using histological and carbon-14 dating approaches, that in adult humans new neurons integrate in the striatum, which is adjacent to this neurogenic niche. The neuronal turnover in the striatum appears restricted to interneurons, and postnatally generated striatal neurons are preferentially depleted in patients with Huntington’s disease. Our findings demonstrate a unique pattern of neurogenesis in the adult human brain.

We here report that neuroblasts are not restricted to the lateral ventricle wall in humans but that they are also present in the adjacent striatum. Retrospective birth dating revealed continuous generation of striatal interneurons in humans. In Huntington’s disease, a neurodegenerative disease affecting striatal neurons (), we find that postnatally generated neurons are absent in advanced stages of the disease. This identifies a unique pattern of adult neurogenesis in humans.

It may appear intuitive that hippocampal neurogenesis has been retained during human evolution, to provide cognitive adaptability, and that olfactory bulb neurogenesis has decreased with the reduced dependence on olfaction in humans compared to our predecessors. However, neuronal precursor cells (neuroblasts) are generated not only in the hippocampus but also in the lateral ventricle wall in adult humans, the site of origin of olfactory bulb neurons in other mammals. The extent and dynamics of neuroblast generation in humans in these two regions are remarkably similar, with a dramatic decline during the first postnatal months, followed by sustained generation, decreasing slowly with age (). The difference between humans and other mammals is thus not the pattern of neuroblast generation but that the neuroblasts generated in the lateral ventricle wall neurogenic niche do not migrate to the olfactory bulb. The fate of neuroblasts born in the human lateral ventricle wall has been unknown.

Whether adult neurogenesis has decreased with evolution has long been a topic of debate (). Humans appear unique among mammals in that there is no detectable adult olfactory bulb neurogenesis (). However, there is substantial hippocampal neurogenesis, with comparable neuronal turnover rates in middle-aged humans and mice (). A larger fraction of hippocampal neurons are subject to exchange in humans than in mice, and adult hippocampal neurogenesis shows a much less dramatic decline with aging in humans compared to mice ().

The generation of new neurons in the adult brain serves to maintain a pool of neurons with unique properties, present for a limited time after their birth, which enable specific types of neural processing (). Adult neurogenesis is important for pattern separation in memory formation and odor discrimination in rodents (), and alterations in adult neurogenesis are implicated in psychiatric disease in humans ().

Interestingly, theC levels in genomic DNA of neurons from grade 2 and 3 Huntington’s disease patients corresponded to the time before the onset of the nuclear bomb tests in 1955 (white dots in Figure 7 C), indicating an absence of postnatally generated neurons. Two subjects with grade 1 Huntington’s disease (gray dots in Figure 7 C), showed slightly elevatedC levels relative to their time of birth. Striatal neurons of patients with Huntington’s disease had significantly lower turnover rates compared to nonaffected age-matched subjects born in the same time frame ( Figure 7 D and Table S5 ). In line with this, we found only 1 out of 786 analyzed striatal neurons devoid of lipofuscin in subjects with Huntington’s disease ( Table S7 ). Individual turnover rates from nonneuronal/nonoligodendrocyte lineage cells were not significantly different between Huntington’s disease patients and nonaffected subjects ( Figures 7 E and 7F), showing that the depletion of adult-born cells was specific to neurons and oligodendrocyte lineage cells in Huntington’s disease.

Huntington’s disease is a neurodegenerative disorder that primarily affects the striatum. However, impaired adult hippocampal neurogenesis has been shown in patients and in transgenic rodent models of Huntington’s disease () and reduced adult olfactory bulb neurogenesis has been detected in a transgenic line of Huntington’s disease mice (), despite the maintenance of the precursor and stem cell pools (). Another study reported increased cell proliferation in the subventricular zone of Huntington’s disease patients (). Therefore, we wanted to determine whether cell turnover dynamics might be affected in the striatum of Huntington’s disease patients. Neuronal, oligodendrocyte lineage and nonneuronal/nonoligodendrocyte lineage cell nuclei were isolated from the postmortem striatum of Huntington’s disease patients by flow cytometry. TheC concentration in genomic DNA from neurons (n = 11 individuals), oligodendrocyte lineage (n = 9), and nonneuronal/nonoligodendrocyte lineage cells (n = 8) was measured (C data are given in Table S5 ). TheC concentration in genomic DNA of oligodendrocyte lineage cells corresponded to time points after the birth of the individuals ( Figure 7 A), but oligodendrocyte lineage cells had significantly lower turnover rates compared to those of healthy age-matched subjects ( Figure 7 B).

(A–F) Cell turnover in the striatum of patients affected by Huntington’s disease. Grey dots indicate individually measuredC concentrations in the genomic DNA of nuclei isolated from the striatum of individuals with grade 1 Huntington’s disease. White dots showC concentrations in the genomic DNA of striatal nuclei from individuals with grade 2 or grade 3 Huntington’s disease. TheC concentration in genomic DNA from oligodendrocyte lineage cells, defined by SOX10 expression, corresponds to time points after the birth of each individual (A). The individual turnover rates from oligodendrocyte lineage cells are significantly lower in individuals with Huntington’s disease (HD) compared to age-matched nonaffected subjects (B).C concentrations in genomic DNA from neuronal nuclei isolated from the striatum of individuals with grade 1 Huntington’s disease are slightly above atmosphericC concentrations at birth, whereasC concentrations in genomic DNA from neuronal nuclei isolated from the striatum of individuals with grade 2 or 3 Huntington’s disease are not significantly different from atmosphericC concentrations at birth (C). The individual turnover rates from striatal neurons are significantly lower in individuals with Huntington’s disease compared to nonaffected age-matched controls (D). TheC concentrations of genomic DNA from nonneuronal/nonoligodendrocyte lineage cells, defined by the absence of NeuN and SOX10 labeling, demonstrate postnatal cell turnover in the striatum of patients affected by Huntington’s disease (E). The individual turnover rates from nonneuronal/nonoligodendrocyte lineage cells do not significantly differ between individuals with Huntington’s disease and nonaffected subjects (F).p < 0.05, Mann-Whitney test for equal medians. Individual turnover rate estimations are based on scenario A (see Extended Experimental Procedures ) to allow comparing healthy subjects with Huntington’s disease patients in spite of the difference in the respective renewing fractions. This results in an underestimation of the turnover rate in the healthy donors compared to the turnover rate in the renewing fraction (scenario 2POP). Error bars in (A), (C), and (E) indicate two standard deviations inC concentration in the respective DNA sample. See also Table S6

The most accurate model for oligodendrocyte lineage cells was a scenario with a high initial turnover rate and lower replacement rate of older cells, suggesting the existence of a subset of short-lived cells ( Table S6 ). This fraction of short-lived cells may correspond to immature oligodendrocyte progenitor cells, since approximately 23% of the SOX10+ nuclei were positive for the mature oligodendrocyte marker APC, indicating that the majority of the isolated oligodendrocyte lineage cells were progenitors. For the nonneuronal/nonoligodendrocyte lineage cells (NeuN-/SOX10−), as for the subset containing all nonneuronal cells (NeuN-), models that allowed one compartment turning over constantly and one nonrenewing population, fitted the data best ( Table S6 ). Individual turnover estimates suggested a decline in striatal nonneuronal cell turnover during aging ( Figures 6 D–6F).

Next, we assessed the turnover dynamics of nonneuronal cells (NeuN-), oligodendrocyte lineage cells (SOX10+), and nonneuronal/nonoligodendrocyte lineage cells (NeuN-/SOX10-) in the lateral wall of the lateral ventricle and in the striatum ( Figures 6 and S5 ). TheC concentration in genomic DNA of all of these cell populations corresponded to time points after the birth of the individuals ( Figures 6 A–6C), establishing turnover of nonneuronal cells.

(E and F) Gene expression analysis of flow cytometry-isolated nuclei shows high expression of marker genes for oligodendrocyte lineage cells (SOX10, CNP, PLP1) in the SOX10+ fraction (E). In contrast, marker genes for astrocytes (GFAP) and hematopoietic cells and microglia (CD45) are highly expressed in the NeuN-/SOX10− sorted nuclei (F).

(D) Immunofluorescence analysis of flow cytometry isolated NeuN-nuclei shows a high percentage of nuclei positive for the oligodendrocyte lineage marker SOX10, whereas neuronal marker HuD, medium spiny neuron marker DARPP32 and interneuron markers calretinin (CR) and ChAT are only expressed in a very small fraction of nuclei and/or attached cytoplasm.

Error bars in (A)−(C) indicate two standard deviations inC concentration in the respective DNA sample. See also Figure S5 and Table S6

(D–F) Individual turnover rates for oligodendrocyte lineage cells (D), nonneuronal/ nonoligodendrocyte lineage cells (E), and all nonneuronal cells (F) computed on the basis of individual data fitting. Individual turnover rates are sensitive to deviations in measuredC and values <0.001 (n = 2) were excluded from the plots, but the full data are given in Table S5 . All individual turnover rates estimates are based on the 2POP scenario.

(A–C) The 14 C concentration in genomic DNA from oligodendrocyte lineage cells (SOX10+) (A), from nonneuronal/nonoligodendrocyte lineage cells (NeuN-/SOX10−) (B), and from all nonneuronal cells (NeuN−) (C), corresponds to time points well after the birth of each individual in the lateral ventricle wall (black dots) and in the striatum (white dots).

C levels in medium spiny neuron DNA corresponded to the time around the birth for most of the individuals ( Figure 5 C), showing that this subtype of striatal neurons is probably not renewed postnatally to a significant level. This finding is in line with the presence of the age pigment lipofuscin in all medium spiny neurons as well as the lack of colocalization of DARPP32 with neuroblast markers or IdU ( Figures 1 and 2 ). In contrast, theC concentration in genomic DNA from interneurons corresponded to time points after the birth of the individuals for the majority of the analyzed subjects ( Figure 5 D), demonstrating postnatal generation of striatal interneurons. Modeling indicates that the striatal neuronal turnover occurs within the interneuron fraction ( Table S6 ).

Given that a large proportion of striatal neurons are not renewed postnatally, we next wanted to identify the subset of striatal neurons that is subject to exchange. The medium spiny projection neurons make up 75%–80% of the striatal neurons, and the four subtypes of interneurons together represent 20%–25% of the striatal neurons (). However, it is not feasible to separately carbon date each of these five neuronal populations with the current sensitivity of accelerator mass spectrometry. For this reason, we aimed at independently carbon dating medium spiny neurons and interneurons. It is challenging to isolate these populations at high purity and therefore not possible to obtain robust quantitative estimates; however, it can point to which neuronal fraction the turnover is in. Striatal nuclei were incubated with antibodies against the neuron-specific nuclear epitope NeuN and the medium spiny neuron marker DARPP32. Nuclei of medium spiny neurons (NeuN+/DARPP32+), interneurons (NeuN+/DARPP32−) and nonneuronal cells (NeuN-) were isolated by flow cytometry ( Figure 5 and Figure S4 ). TheC concentration in genomic DNA from medium spiny neurons (n = 26 individuals), interneurons (n = 11), and nonneuronal cells (n = 18) was measured by accelerator mass spectrometry (C data are given in Table S5 ).

(E) Antibodies to NeuN label all neurons in the striatum, whereas DARPP32 expression is specific to medium spiny neurons, which constitute the vast majority of the striatal neurons. The minority population of striatal interneurons is defined by the presence of NeuN and the absence of DARPP32 (arrows). Scale bar, 20 μm.

(D) Immunofluorescence analysis of flow cytometry-isolated NeuN+/DARPP32− nuclei shows a high percentage of nuclei and/or attached cytoplasm positive for the neuronal marker HuD and for the interneuron markers calretinin (CR) and choline acetyltransferase (ChAT). In contrast, oligodendrocyte lineage marker SOX10 and medium spiny neuron marker DARPP32 are expressed in a small fraction of nuclei only.

(B) Immunofluorescence analysis of flow cytometry-isolated NeuN+/DARPP32+ nuclei shows a high percentage of nuclei positive for the neuronal marker HuD and for the medium spiny neuron marker DARPP32. In contrast, oligodendrocyte lineage marker SOX10 and interneuron markers calretinin (CR) and choline acetyltransferase (ChAT) are expressed only in a small percentage of nuclei and/or attached cytoplasm.

(D) The 14 C concentration in genomic DNA from striatal interneurons corresponds to time points after the birth of most individuals, demonstrating interneuron renewal throughout life. Error bars indicate two standard deviations in 14 C concentration in the respective DNA sample.

(C) 14 C concentrations in genomic DNA from striatal medium spiny neuron nuclei (NeuN+/DARPP32+) are not significantly different from atmospheric 14 C concentrations at birth for the vast majority of the analyzed individuals.

(A and B) Cell nuclei were isolated from the human postmortem striatum and left unstained (A) or incubated with antibodies against NeuN and against the medium spiny neuron marker DARPP32 (B). The interneuron (NeuN+/DARPP32−), medium spiny neuron (NeuN+/DARPP32+), and nonneuronal populations (NeuN−) were isolated by flow cytometry. The sorting gates are indicated.

First, we analyzed theC concentration in neuronal genomic DNA in the lateral wall of the lateral ventricle and in the striatum ( Figure 4 C). For the majority of the analyzed subjects, theC concentration in neuronal genomic DNA corresponded to the concentration in the atmosphere after the birth of the individual, showing the postnatal generation of striatal neurons. In contrast, cortical, cerebellar and olfactory bulb neurons are not renewed postnatally to a detectable level in humans, andC levels in their DNA correspond to the time around the birth of the individual ( Figure S3 ) (). Individuals born before the nuclear bomb tests had lowerC levels in striatal neuron DNA than at any time after 1955, establishing that although some neurons are generated postnatally, a large majority of striatal neurons are not exchanged after birth. Mathematical modeling ofC data allows a comprehensive analysis of cell turnover (). By fitting several mathematical models to the data, the cell renewal rate and the fraction of cells showing turnover were estimated (see Table S6 and Extended Experimental Procedures ). The best model was a scenario in which a subpopulation of the neurons is renewing, whereas the majority is not. The size of the cycling neuronal population was 25% (95% confidence interval: 6%–51%). Individual estimates of turnover rates showed a modest decline in turnover over age within the cycling population ( Figure 4 D). The median turnover rate of neurons within the renewing fraction was 2.7% per year in adulthood, which is not significantly different (p = 0.7, two-tailed Mann-Whitney test) compared to the turnover rate of neurons within the renewing fraction in the adult human hippocampus ().

To specifically birth date different cell types in the lateral ventricle wall and striatum, nuclei were incubated with antibodies against the neuron-specific nuclear epitope NeuN and the oligodendrocyte lineage marker SOX10. Neuronal, oligodendrocyte lineage and nonneuronal/nonoligodendrocyte lineage nuclei were isolated by flow cytometry ( Figures 4 and S3 ) (). Reanalysis by flow cytometry and analysis of mRNA expression indicated the specificity of the isolation ( Figure S3 ). TheC concentration in genomic DNA from neurons (n = 30 individuals), oligodendrocyte lineage cells (n = 28), and nonneuronal/nonoligodendrocyte lineage cells (n = 26) was measured in subjects from 3 to 79 years of age (C data are given in Table S5 ).

(D) 14 C concentrations in genomic DNA from neuronal nuclei isolated from the cerebellum and from the occipital cortex do not significantly differ from atmospheric 14 C concentrations at birth.

(B) Gene expression analysis of flow cytometry-isolated nuclei shows high expression of neuronal markers NeuN and MAP2 in the NeuN-positive fraction. In contrast, marker genes for oligodendrocyte lineage cells (SOX10, CNP, PLP1) and marker genes for astrocytes (GFAP) and hematopoietic cells and microglia (CD45) are not expressed in NeuN+ sorted nuclei (C). The antibody to NeuN labels neurons in the lateral wall of the lateral ventricle and in the striatum, as confirmed by the expression of the neuronal marker MAP2. Scale bar, 20 μm. Error bars indicate two standard deviations in 14 C concentration in the respective DNA sample.

(D) Individual turnover rates for neuronal cells computed on the basis of individual data fitting according to the 2POP scenario (see Extended Experimental Procedures ). Individual turnover rates are sensitive to deviations in measuredC, especially for young individuals due to the shallow slope of theC curve, and values <0.001 (n = 3) or >1.00 (n = 8) were excluded from the plot, but the full data are given in Table S5 . The individual turnover rates for adult subjects were not significantly different between neurons from the lateral ventricle wall and from the striatum (p = 0.9, Mann-Whitney test) but significantly higher than the turnover rates observed in the cortex or cerebellum (p < 0.05, Mann-Whitney test).

(C) 14 C concentrations in the lateral ventricle wall and striatal neuron genomic DNA correspond to a time after the date of birth of the individual for the majority of the analyzed subjects, demonstrating neurogenesis throughout life. Error bars indicate two standard deviations in 14 C concentration in the respective DNA sample.

(A and B) Isolation of nuclei from neurons, oligodendrocyte lineage and nonneuronal/nonoligodendrocyte lineage cells from the lateral ventricle wall and from the striatum. Cell nuclei were isolated from human postmortem tissue and left unstained (A) or incubated with antibodies against the neuron-specific epitope (NeuN) and the oligodendrocyte lineage marker SOX10 (B). The neuronal (NeuN+), oligodendrocyte lineage (SOX10+) and nonneuronal/nonoligodendrocyte lineage populations (NeuN-/SOX10−) were isolated by flow cytometry. The sorting gates are indicated.

We isolated cell nuclei from the human postmortem lateral wall of the lateral ventricle and from the striatum by gradient centrifugation. Analysis of theC concentration in their genomic DNA by accelerator mass spectrometry revealed levels corresponding to time points after the birth of the individuals, showing that there is postnatal cell turnover in the human striatum and in the lateral wall of the lateral ventricle ( Figure 3 B and Table S5 ). There was no significant difference in cell turnover in the striatum and in the lateral ventricle wall (p = 0.71, two-tailed Mann-Whitney test). In line with a population comprising several cell types, subpopulation dynamics analysis pointed to a heterogeneous group of cells, with some subpopulations having high turnover rates, others low ones, and a large proportion of cells not being exchanged at all postnatally ( Figure 3 C).

It is difficult to estimate the dynamics of neurogenesis based on the incorporation of labeled nucleotides. This is especially true in humans, as the access to tissue is very limited and it is inevitable that the subjects receive different doses at different times prior to their death. In order to explore turnover dynamics of cells in humans, we have developed a method to retrospectively birth date cells, which is based on the integration of nuclear-bomb-test-derivedC in DNA of proliferating cells ( Figure 3 A) ().

(C) In line with a tissue composed of many different cell types, subpopulation dynamics analysis indicates that striatal cells form a heterogeneous group, some fractions having high turnover rates and some having very low ones. The gray area represents the range of acceptable values. The resolution of this type of analysis does not allow differentiating between turnover rates above 10%.

(B) The 14 C concentrations in genomic DNA from cell nuclei isolated from the lateral wall of the lateral ventricle (black dots) and from the striatum (white dots) demonstrate postnatal cell turnover in subjects born before and after the bomb spike. Error bars indicate two standard deviations in 14 C concentration in the respective DNA sample.

(A) Schematic illustration ofC concentration measurements in genomic DNA. The black line shows the atmosphericC concentration over time. Individually measuredC concentrations in human genomic DNA are plotted at the time of the subject’s birth (vertical lines), before (blue symbols) or after theC bomb spike (red symbols). Data points above the bomb curve for subjects born before the bomb peak and below the bomb curve for subjects born after the nuclear tests indicate cell turnover. Data points on the atmospheric curve show the absence of turnover. Data from, and

Thymidine analogs incorporated into the DNA of dividing cells can be detected in their progeny, allowing for the identification of newly generated cells. We analyzed postmortem tissue from the striatum, hippocampus, and cortex from cancer patients who received iododeoxyuridine (IdU) for radiosensitization (see Table S4 for information on the patients). In patients receiving the lowest IdU concentrations, we failed to detect any IdU-labeled cells, neuronal or nonneuronal, in the brain. However, in all individuals in whom we found IdU-labeled nonneuronal cells (n = 4 subjects, age 20–71), we also detected IdU-labeled cells in the striatum coexpressing the neuronal markers NeuN, MAP2, and/or calretinin ( Figures 2 C–2E). IdU-labeled neurons were found in both the caudate nucleus and the putamen. The nuclei of IdU-positive cells were uniformly labeled, and not in a punctuate pattern, the later being associated with DNA repair. IdU-positive neurons were also detected in the dentate gyrus of the hippocampus in the same subjects ( Figure S2 ), but not in the cerebral cortex, in line with previous studies ().

(A) Newly generated neurons can be detected in the adult human dentate gyrus of the hippocampus in patients previously receiving IdU. Confocal images with orthogonal projections show an IdU-labeled neuron expressing MAP2.

We examined lipofuscin accumulation in the five major neuronal subtypes of the human striatum. We found that 4.7% of the calretinin-expressing interneurons were devoid of lipofuscin and a larger fraction (16.8%) had low lipofuscin content in adult humans ( Figures 2 A and 2B ). A similar fraction of NPY-positive neurons had a low lipofuscin content (19.5%), but only very few (0.6%) were completely devoid of lipofuscin. The other neuronal subclasses were dominated by cells with high lipofuscin content, and we failed to find any such cells devoid of lipofuscin. It is not possible to deduce the age of a cell based on its lipofuscin content. However, we did not find any lipofuscin in striatal neurons in a 9-month-old subject, whereas >96% of striatal neurons contained lipofuscin in a 2.8- and a 6-year-old individual (as well as in all older studied subjects), suggesting that it may take a few years for newborn striatal neurons to accumulate lipofuscin ( Table S3 ).

(C–E) Newly generated cells can be detected in the adult human striatum in patients previously receiving the thymidine analog IdU. Confocal images with orthogonal projections showing IdU-labeled neuronal nuclei in the striatum of two subjects aged 20 and 41. Neurons are identified by the expression of NeuN, MAP2 and/or calretinin (CR).

(B) Confocal images with orthogonal views demonstrate the absence of lipofuscin granules in a calretinin-positive neuron (arrow) while other cells have autofluorescent lipofuscin pigments in their cytoplasm, visible in all channels (star).

(A) Lipofuscin quantification by confocal microscopy in the five main subtypes of striatal neurons. DARPP32, dopamine- and cAMP-regulated neuronal phosphoprotein; CR, calretinin; ChAT, Choline acetyltransferase; PV, parvalbumin; NPY, neuropeptide Y. Data are shown as mean values for three donors aged between 21 and 26. Error bars represent standard deviation. A minimum of 200 neurons per subtype and per subject were analyzed.

Over time, cells accumulate the age pigment lipofuscin, consisting of autofluorescent matrix and lipid droplets (). There are no definitive molecular markers for neuroblasts, and some nonrenewing neurons have been reported to express DCX () or PSA-NCAM (). However, most DCX-positive cells in the striatum were devoid of lipofuscin ( Figure 1 F), suggesting that they are indeed newly generated cells, and those that did have lipofuscin contained only a few granules. The vast majority of the hippocampal neurons of the granular zone of the dentate gyrus also lack lipofuscin in adult humans ( Table S3 ).

The five main neuronal subtypes in the striatum can be identified by the markers dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP32), calretinin, choline acetyltransferase, parvalbumin, and neuropeptide Y. DCX most commonly colocalized with calretinin and more rarely with neuropeptide Y ( Figures 1 D and 1E, Table S2 ), whereas we did not find any colocalization with markers for the other subtypes. We did not detect DCX-positive cells with the apoptotic marker activated caspase-3 in the subventricular zone or striatum. In contrast, cleaved caspase-3 was detected in some DCX-positive cells in the subventricular zone of subjects with Huntington’s disease ( Figures S1 B–S1E).

Immunohistochemistry revealed DCX-positive putative neuroblasts in the adult human dentate gyrus of the hippocampus and in the subventricular zone of the lateral wall of the lateral ventricle ( Figure S1 ), in line with previous studies (). DCX-positive cells were also present in the caudate nucleus and the putamen of striatum ( Figures 1 C–1F). The morphology of the DCX-positive cells in the striatum varied from rounded without processes, elongated with few processes to highly branched ( Figures 1 C–1F), similar to the neuroblasts in the subventricular zone and hippocampus (). The DCX-positive cells in the striatum were most often found as single isolated cells, and there were no apparent streams of potentially migratory cells from the subventricular zone. Most of the DCX-positive cells in the dentate gyrus, subventricular zone, and striatum were also positive for PSA-NCAM and the neuronal marker NeuN, but they were invariably negative for the astrocytic marker GFAP ( Figure 1 C and S1 Table S2 ). The presence of the mature neuronal marker NeuN in many DCX-positive neurons does not necessarily imply that they are not newborn; in nonhuman primates, adult-born neurons in the dentate gyrus maintain DCX for at least 6 months and coexpress NeuN (), suggesting that they may retain a juvenile state for a substantial period of time.

Western blot analysis of the human postmortem hippocampus, striatum, and cerebellum from subjects aged 21 to 68 years showed that DCX and polysialylated neural cell adhesion molecule (PSA-NCAM), another marker associated with neuroblasts, were as abundant in the striatum as in the hippocampus, and low to undetectable in the adult cerebellum ( Figure 1 B). This is in line with a previous study demonstrating DCX and PSA-NCAM by western blot in the adult human striatum, with similar levels in the caudate nucleus and putamen ().

The finding that neurons are not added in the olfactory bulb of adult humans (), in spite of the generation of neuronal precursors in the subventricular zone (), posed the question of whether neuroblasts may migrate to another location close to the ventricle. Analysis of transcriptome data available from a large number of developing and adult human brains () demonstrated that the expression of doublecortin (DCX), a commonly used neuroblast marker, was at least as high in the adult human striatum as in the hippocampus ( Figure 1 A and Table S1 available online). Only background levels were detected in the nonneurogenic adult cerebellum ( Figure 1 A, Table S1 ). When comparing DCX expression levels in the striatum with other brain regions close to the lateral ventricle, high DCX mRNA levels were specific to the striatum in the data fromas well as in an additional human transcriptome data set ( Figure S1 F). DCX transcript levels in the human hippocampus correlate closely with the number of neuroblasts at different ages () and the number of DCX-positive cells in the hippocampus in turn correlates with the number of newly generated neurons ().

(F) Comparison of z-score-normalized DCX mRNA expression in the human striatum, other regions close to the lateral ventricle and in the nonneurogenic cortex. The subjects are aged 21 to 57, n = 6 (data from the Allen brain atlas). Error bars show standard deviation between subjects.

(A–E) Two different DCX antisera label the same cells (A). The guinea-pig-derived antiserum was used in the western blot in Figure 1 B. The majority of DCX-positive cells coexpress the neuroblast marker PSA-NCAM and all of them are negative for the apoptotic marker cleaved caspase 3 (B–D). Apoptotic DCX-positive cells are seen in the subventricular zone (SVZ) of patients affected by Huntington’s disease (HD) (E). Scale bars, 20 μm.

Neuroblast Marker Expression in the Dentate Gyrus of the Hippocampus, in the Subventricular Zone and in the Striatum, Related to Figure 1

(C–F) Confocal microscopy of DCX-positive cells in the striatum. The majority of the DCX-positive cells express the mature neuronal marker NeuN but all of them lack expression of the astrocytic marker GFAP (C). Most of the DCX-positive cells also express PSA-NCAM (D). DCX occasionally colocalizes with neuronal markers calretinin (CR) (D) and NPY (E). Most DCX-positive cells have little or no lipofuscin (arrow), whereas the majority of DCX-negative cells contain lipofuscin pigments (stars) (F). Autofluorescent lipofuscin pigments are not visible in all channels because an antiautofluorescence treatment was applied. Scale bars, 20 μm for (C)–(E) and 10 μm for (F). Cell nuclei are labeled with DAPI and appear blue.

(B) Western blot analysis of DCX, PSA-NCAM and β-actin in the hippocampus (H), striatum (S), and cerebellum (C) of human subjects of different ages.

(A) DCX expression in the striatum (green), hippocampus (red), and cerebellum (blue) across the human lifespan. Data from. The striatal area used for the transcriptome analysis comprises the caudate nucleus (lateral ventricle wall included), the nucleus accumbens, and the putamen. See also Table S1

Discussion

Spalding et al., 2013 Spalding K.L.

Bergmann O.

Alkass K.

Bernard S.

Salehpour M.

Huttner H.B.

Boström E.

Westerlund I.

Vial C.

Buchholz B.A.

et al. Dynamics of hippocampal neurogenesis in adult humans. New neurons are continuously added in the olfactory bulb and hippocampus in most mammals. Humans show substantial hippocampal neurogenesis (), but are unique in that there is no detectable addition of neurons in the olfactory bulb. However, the density of neuroblasts is very similar in the subventricular zone and the dentate gyrus of the hippocampus. We report that cells expressing the neuroblast markers DCX and PSA-NCAM are present not only in the adult human subventricular zone, but also in the adjacent striatum. IdU in striatal interneurons indicate the generation of this cell type in adult humans and retrospective birth dating of striatal neurons confirms the generation of interneurons. We furthermore report that adult-generated striatal neurons are preferentially depleted in Huntington’s disease. The identification of continuous generation of striatal interneurons identifies a unique pattern of adult neurogenesis in humans.

14C monitor DNA synthesis during cell proliferation. However, chromosomal damage and repair can result in DNA synthesis. Since DNA damage and repair occur almost exclusively during the cell cycle, this would not affect the outcome of our analysis. DNA repair is thought to be very limited in postmitotic cells, and well below the detection limit of carbon dating ( Spalding et al., 2005 Spalding K.L.

Bhardwaj R.D.

Buchholz B.A.

Druid H.

Frisén J. Retrospective birth dating of cells in humans. 14C in DNA over several decades in cerebellar, cortical, or olfactory bulb neurons in humans ( Bergmann et al., 2012 Bergmann O.

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et al. The age of olfactory bulb neurons in humans. Bhardwaj et al., 2006 Bhardwaj R.D.

Curtis M.A.

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Frisén J. Neocortical neurogenesis in humans is restricted to development. 14C concentrations in genomic DNA from cortical neurons after ischemic stroke, which induces massive DNA damage, are not significantly different from that present in the atmosphere at the time of birth of the individuals (unpublished data). Mathematical modeling established that the integration of 14C in DNA is limited to a subpopulation of neurons, and if the DNA synthesis would be due to DNA damage and repair, these neurons would have to have exchanged their entire genomes, which appears highly unlikely. The finding that neurons in a distinct striatal subpopulation express neuroblast markers, lack age pigment, and are labeled with IdU and 14C, together lend strong support to the conclusion that striatal neurons are generated in adulthood in humans. It is important to consider alternative interpretations of our results. Both the integration of IdU andC monitor DNA synthesis during cell proliferation. However, chromosomal damage and repair can result in DNA synthesis. Since DNA damage and repair occur almost exclusively during the cell cycle, this would not affect the outcome of our analysis. DNA repair is thought to be very limited in postmitotic cells, and well below the detection limit of carbon dating (). We have not been able to detect any incorporation ofC in DNA over several decades in cerebellar, cortical, or olfactory bulb neurons in humans (). EvenC concentrations in genomic DNA from cortical neurons after ischemic stroke, which induces massive DNA damage, are not significantly different from that present in the atmosphere at the time of birth of the individuals (unpublished data). Mathematical modeling established that the integration ofC in DNA is limited to a subpopulation of neurons, and if the DNA synthesis would be due to DNA damage and repair, these neurons would have to have exchanged their entire genomes, which appears highly unlikely. The finding that neurons in a distinct striatal subpopulation express neuroblast markers, lack age pigment, and are labeled with IdU andC, together lend strong support to the conclusion that striatal neurons are generated in adulthood in humans.

Tepper and Bolam, 2004 Tepper J.M.

Bolam J.P. Functional diversity and specificity of neostriatal interneurons. Another major difference between rodents and humans may be the neuronal subtypes generated in the subventricular zone. Calretinin-expressing interneurons are much rarer in rodents, constituting less than 1% of striatal neurons, compared to humans, in whom 10% of striatal interneurons are calretinin positive. The function of this neuronal subclass is essentially unknown, to a large extent due to its paucity in animals amenable to experimental manipulation. Similar to their human counterpart, rodent calretinin-positive interneurons are of medium size and aspiny. However, only a very limited description of their axonal arborization is available and their electrophysiological profile remains unknown ().

Zuccato et al., 2010 Zuccato C.

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Molina-Calavita M.

Keryer G.

Zala D.

Charrin B.C.

Dietrich P.

Volvert M.L.

Guillemot F.

Dragatsis I.

et al. Huntingtin is required for mitotic spindle orientation and mammalian neurogenesis. The depletion of adult-born neurons in Huntington’s disease may be due to reduced generation of neurons and/or preferential degeneration of adult-born neurons. Huntington’s disease is thought to be the result of both loss of function of the normal Huntingtin protein and toxic effects of the modified protein (). Huntingtin has been implicated in both embryonic and adult neurogenesis (), and it is possible that failing striatal neurogenesis contributes to the depletion of adult generated neurons. However, since the subjects with early stage disease showed a more modest depletion of adult-born neurons than at the advanced stages ( Figure 7 ), adult-born neurons may be lost with progression of the disease, although the number of subjects available for analysis was limited.

We can currently only speculate about the potential function of continuous striatal neurogenesis in humans, and the functional integration of the new neurons into existing neuronal circuits as well as their effects remain to be investigated. The striatum was first associated with motor control, but it is today well-established that this region also is important for many cognitive functions. Huntington’s disease results in motor, cognitive and psychiatric symptoms, with the motor symptoms often being preceded by cognitive impairment. The selective depletion of adult-generated neurons in Huntington’s disease indicates that the effect of losing such neurons may be found within the symptoms that these patients display. However, medium spiny neurons account for most of the neuronal loss, and most of the symptomatology is likely explained by this.