Deficient progenitor proliferation in HD-hCOs

To study early corticogenesis in HD patients, we used human iPSC lines derived from the dermal fibroblasts of two HD patients (CAG 55 and 59) to generate HD-hCOs (Fig. 1A). Healthy hCOs were derived from hiPSCs (CAG 19; healthy sibling) and the embryonic stem cell line H9 was used as a control (CTR) (Fig. 1A). We performed a rigorous growth trajectory analysis of hCOs for 60 days (Fig. S1A). The growth curve showed that CTR-hCOs expanded faster than HD-hCOs after approximately 20 days (Fig. S1A). Notably, we observed that the neural rosettes in the CTR group were larger and richer than those in the HD group (Fig. 1B, S1B).

Fig. 1: Impaired neural tubes present in HD hCO.

A The schematic of the construction of hCOs and comparing strategy. CTR, control Group, including H9 and CAG 19; HD, Huntington’s Group, including CAG 55 and 59. B Representative bright-field images of CTR and HD hCOs on Day 25 showed multiple typical neural tube-like structure presented in hCOs (yellow arrows, neural tubes). C Immunostaining with TUJ1 and SOX2 antibodies outlined the neural tubes of hCOs on Day 30, 47 and 60 (yellow dashed lines, the borders of neural tubes in hCOs). D Comparing the ratio of neural tube (n = 6) and TUJ1+ coverage area (n = 6) of HD hCOs on Day 30, 47 and 60 with CTR (CAG 55 and 59 vs. H9 and CAG 19). Data, mean ± s.e.m. One-way ANOVA. *P < 0.05; **P < 0.01. E Representative images and counting of Ki67 (n = 6) antibody immunostaining in HD and CTR hCO (CAG 55 and 59 vs. H9 and CAG 19) and human fetal brain tissue (n = 1) (yellow dashed lines, the borders of neural tube). Comparing the ratio of Ki67+ (n = 6) cells on Day 30, 47 and 60 with CTR (CAG 55 and 59 vs. H9 and CAG 19). Data, mean ± s.e.m. One-way ANOVA. *P < 0.05. F Representative images and counting of Ki67 (n = 6) antibody immunostaining in the apical and basal side of CTR and HD hCO (CAG 55 and 59 vs. H9 and CAG 19) on Day 47 and human fetal brain tissue (n = 1) (yellow dashed lines, the borders of apical surface and basal surface; yellow arrows, typical Ki67+ cells). G Schematic of CTR/HD chimerism and chimeric rosette. H Representative images of Ki67 antibody immunostaining and counting Ki67+ cells (n = 5) in chimeric organoids (CAG 55 and 59 vs. H9 and CAG 19). Data, mean ± s.e.m. Student’s t-test. **P < 0.01. I–J Representative images of Arl13b antibody immunostaining (I) and calibrating Arl13b+ cilia length (n = 6) and density (n = 6) in the HD and CTR hCOs (CAG 55 and 59 vs. H9 and CAG 19) on Day 47(J). Data, mean ± s.e.m. Student’s t-test. **P < 0.01. K Immunostaining of PAX6 and TBR2 antibodies in HD and CTR hCOs on Day 30, 47 and 60 (dashed lines, boundaries of the VZ-like zones). L Comparing the percentage of PAX6-negative, TBR2-positive cells (PAX6+/TBR2+) over PAX6-positive, TBR2-negative (PAX6+/TBR2-) progenitors (n = 6) in HD and CTR hCOs (CAG 55 and 59 vs. H9 and CAG 19). Data, mean ± s.e.m. One-way ANOVA. *P < 0.05; ns, nonsignificant.

Brain organoids, a model of human brain development in vitro, comprise a population of neuroepithelial cells (NEs) assembled into large neural rosettes during the early period, representing the origin of neurogenesis [15, 20]. Staining with the apical complex (AC) markers N-cadherin (NCAD), β-catenin, and ZO-1 revealed the formation of junctional complexes in the apical domain (Fig. S1C), demonstrating apicobasal polarity in the neuroepithelium of the rosettes. To investigate HD neurogenesis, we selected organoids on days 30, 47, and 60, and stained the neural tube with antibodies against SOX2, an early neuroepithelial marker, and TUJ1, a pan neural marker (Fig. 1C, S2A). Calibration of the immunostaining results showed that the thickness of the neural tube and coverage of the neural tube-like area in HD-hCOs matched observations in the bright field (Fig. 1D, S2B). The increase in the TUJ1+ neuronal area paralleled the decrease of the neural tube in both the HD and CTR groups, whereas the increase in the CTR group was slower (Fig. 1D).

The balance between proliferation and differentiation of progenitors is a crucial determinant of neural tube size during development [21]. Therefore, we evaluated proliferative NEs using Ki67 and phospho-histone 3 (PH3) antibody immunolabeling (Fig. 1E, S2C). A sharp decline in mitotic progenitors in the HD group compared to the CTR group was observed from day 30 to 47 (Fig. 1E, S2D). TUNEL staining showed that the neural tubes in early HD cortical organoids did not contain more apoptotic cells than those in the CTR group (Fig. S2E). This excluded the possibility that the increase in apoptosis in progenitor cells caused a thinner neural tube in HD-hCOs. Remarkably, more Ki67+ cells were present basally in the VZ zone of the HD-hCOs compared with the CTR group and healthy human fetal cortex (GW9) (Fig. 1F) To exclude variations between different organoids, we constructed two chimeric hCOs that contained a uniform mixture (1:1) of HD and CTR cells, and one line (HD or CTR) was stably transfected with green fluorescent gene (GFP) (Fig. 1G). During monitoring, a large neural tube containing both GFP+ and unlabeled cells was observed (Fig. S3A), and histology further revealed that GFP+ cells were uniformly mixed with the untagged cells (Fig. S3B). However, the HD cells tended to be excluded from the VZ-like area (Fig. S3C). The GFP+ fluorescence intensity indicated that HD cells tended to be distributed basally compared to CTR cells in the VZ (Fig. S3D and S3E). Consistent with the phenotype of single hCOs, the proportion of HD Ki67+ neuroepithelial cells in the same neural tube was lower (Fig. 1H) and tended to be basally distributed (Fig. S3F). These findings indicate that early exhaustion of progenitors might be a common phenomenon in HD-hCOs, contributing to the thinner cortex in the fetal and childhood brain that carry mHTT.

Premature neurogenesis in HD-hCOs

Defects in neural progenitor differentiation and neurogenesis biased toward the neuronal lineage have been observed in the human HD fetal brain at 13 gestational weeks (GW) [6]. We investigated whether HD-hCOs model premature neurogenesis in HD fetuses. Arl13b antibody staining, a marker for cilia, showed that Arl13b-positive cilia were present in the apical domain of the neuroepithelium in cortical organoids, resembling cilia in healthy human fetuses (GW9) (Fig. S3G). Apical cilia in the neural tube of the HD group were longer and denser than those in the CTR group and normal human fetal brains (Fig. 1I, J), reflecting less mitotic activity in the neural tube in HD-hCOs. To determine whether the reduction in mitosis disrupts the status of the apical progenitor (AP) and basal progenitor (BP) populations in HD-hCOs, we stained hCOs with antibodies against the AP marker PAX6 and BP marker TBR2 (Fig. 1K). The staining results showed that the ratios of TBR2+PAX6-/TBR2-PAX6+ in the HD group were higher than those in the CTR group (Fig. 1L), indicating that early lineage specification of the neural progenitors was preceded in HD-hCOs, which is consistent with the observations in HD fetuses and embryos of HD mice [6].

To understand the mechanisms underlying neurodevelopment in HD cortical organoids, we analyzed the global transcriptomes of the organoids on days 30 and 60. Differential gene expression analysis between the HD (55 or 59 CAGs) and control (19 CAGs, H9) groups identified 471 differentially expressed genes (DEGs) on day 30 and 1110 DEGs on day 60 (Fig. S4A). Principal component analysis showed a clear separation of HD and CTR hCOs on days 30 and 60 (Fig. S4B). The DEGs included several critical genes relevant to neurodevelopment (Fig. S4C): HES3 [22], NEUROD4 [23], NEUROG1 [24], NKX6.1/2 [25], and WNT5A [25], typical neurogenesis regulators, were upregulated on day 30, whereas FEZF2 [26], which regulates the fate of subcortical projection neurons, was downregulated. Notably, several genes showed opposite differential trends at the two time points, including FAM107A [27] and GSX2 [28], and differences in the earliest transcription factors expressed in neuronal progenitors, including DLX2/5 [29], ARX [30], and ZNF536 [31], which are regulators of neuronal differentiation. The glutamate receptor-related genes GRIK2 [32] and GRM5 [33] and the synaptic transmission-related genes CPLX1 [34], NGEF [35], SYNGR1 [36], and SYNPR [37] were also upregulated on day 60 (Fig. S4C). To further investigate the divergent developmental trajectory between CTR and HD, we compared the similarity of transcriptomic profiles from HD and CTR organoids with published transcriptomic profiles from the human fetal brain in three different cortical regions and distinct developmental periods ranging from 8 post-conception week to 4 months (Fig. S4D). The results showed that on day 60, the highest correlated human brain development stage of HD-hCOs was later than that of the CTR group, similar to the pattern on day 30. Transcriptomics verified premature neurogenesis in HD-hCOs.

Deficiency of cortical projection neurons and laminations in HD hCOs

From the beginning of neurogenesis, newborn neurons radially migrate toward the pia surface, populating the cortical plate (CP) that constitutes the different layers in a nested “inside-out” progression from the deeper cortex to the more superficial cortex [38, 39] (Fig. 2A). To determine whether premature neurogenesis and the deficiency of proliferative progenitors alter the structure of the CP-like region in hCOs, we used TBR1, CTIP2, and SATB2 antibodies, representing the projection neurons in the different cortical layers, to stratify the cortical layers [26, 40,41,42]. TBR1+, CTIP2+, and SATB2+ cells covered most regions outside the VZ in CTR-hCOs, outlining the CP-like regions in hCOs and mimicking the layering of three projection neurons in the human fetal brain (Figs. 2B, S5A and S5B). In contrast, TBR1+, CTIP2+, and SATB2 + cells in HD-hCO were scattered in the CP-like regions of HD-hCOs (Figs. 2C, S5C). The cell count results of hCOs on days 47, 60, and 80 showed that the ratio of TBR1+, CTIP2+, and SATB2+ cells in the HD group was lower than that in the CTR group, and the differences in SATB2+ and TBR1+ cells were striking (there were too few TBR1+ cells in the HD group to count) (Fig. 2D).

Fig. 2: Perturbed cortical laminations and fate specification in cortical neuron subtypes in HD hCOs.

A Schematic of the neuronal marker expression during cortical maturation and neural layering in healthy fetal brain (VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate). B, C Stitched and single channel images of TBR1, CTIP2 and SATB2 antibodies immunostaining in the different layers, including CP, VZ and SVZ regions, of CTR (B) and HD hCOs (C). The magnified images with single channel in the lower panel showed the gradient patterns of TBR1, CTIP2 and SATB2+ cells in the different layers of hCOs (yellow dashed lines, the margins of the VZ and SVZ-like area). The VZ-like area on Day 80 HD hCOs are chaotic (not indicate by a dashed line). D Comparing TBR1+, CTIP2+ or SATB2+ cells in HD hCOs with CTR (n = 8) on Day 47, 60 and 80 (CAG 55 and 59 vs. H9 and CAG 19). Data, mean ± s.e.m. One-way ANOVA. *P < 0.05; **P < 0.01; ns, nonsignificant. E Comparing the distributing tendency of TBR1+, CTIP2+ and SATB2+ neurons (n = 8) in the CP-like of hCOs of HD group with CTR (CAG 55 and 59 vs. H9 and CAG 19). The CP-like is evenly divided into 10 bins, following the apical-to-basal direction. Curves representing the normalized abundance within each bin. The value, marked cells in a bin / total neurons of CP-like. Data, mean ± s.e.m.

Based on another protocol [43], we divided the thickness of the CP into 10 evenly spaced bins to evaluate the laminar patterns of TBR1+, CTIP2+, and SATB2+ cells. In the CTR group, TBR1 and SATB2 exhibited two separate peaks, representing the upper and deep layers, on day 80, and CTIP2+ cells spanned the entire CP (Fig. 2B, E), indicating layer-specific marker expression in the human neocortex [41]. In contrast, TBR1+ cells were barely observed in HD-hCOs on days 47, 60, and 80, and SATB2+ cells displayed more mutually exclusive domains with CTIP2+ cells in the deep layers on day 60 (Fig. 2C, E) than that in CTR group. Assessment of laminar expression patterns in the HD group was challenging on day 80 because of neural tube exhaustion, and SATB2+ cells were scattered in the region of CTIP2+ cells (Fig. 2C, S6A). Collectively, these results revealed that the decrease in cortical projection neurons in the HD group was accompanied by the perturbation of neuronal layering.

Similar to the segregation of CTIP2 and SATB2, including the increase in CTIP2+ and CTIP2+/SATB2+ cells, SATB2+ deep neurons continuously increased in CTR-hCOs, suggesting the post-mitotic fate specification of existing cortical neurons during the establishment of separated laminar expression domains (Fig. 2B, D). These observations are reminiscent of the cortical development of newborn excitatory neurons that co-express the projection specification transcription factors SATB2 and CTIP2, and these markers then segregate entirely by a narrow topographical transition [43, 44]. Neither SATB2+ cells in HD-hCOs increased from 47 to 80 days, nor did SATB2+/CTIP2+ cells increase compared to CTRs (Fig. 2D). The accumulation of CTIP2+ cells and the decrease in CTIP2+/SATB2+ cells in the HD group were dramatic compared to those in the CTR group (Fig. 2C–E). Together, these findings imply that the aberrant CP-like regions in the HD group may be due to the dysregulated fate specification of specific cortical neuron subtypes. The capacity of HD progenitors to differentiate into CTIP2+ and SATB2+ neurons was identical to that of CTRs in the chimeric organoids (Fig. S6B–S6D). However, the decrease in colabelled HD cells revealed impaired fate determination of neuronal subtypes in chimeric organoids (Fig. S6D).

Delayed maturation of postmitotic neurons may contribute to the abnormal accumulation of CTIP2 in HD-hCOs

To determine whether HD neuronal progenitors also entered the mature stage, we used SOX2 and NEUN, mature neuron markers, to stain CTR and HD-hCOs on days 60 and 80. SOX2+ cells were restricted to the VZ-like zones, whereas NEUN+ cells covered the remaining areas in the CTR group. Massive SOX2+ cells ectopically appeared in the outer region of the VZ in HD-hCOs (Fig. S7A). NEUN+ cells were rarer in HD-hCOs than in CTR-hCOs. The increase in SOX2+ cells and decrease in NEUN+ cells in the outer region of the VZ-like zones in the HD group differed from those in the CTR group (Fig. S7B). Notably, the number of ectopic SOX2+ cells in the outer region of the VZ-like zones decreased in a time-dependent manner in HD-hCOs from days 60 to 80. At the same time, the ratio of NEUN+ mature neurons also decreased (Fig. S7A), indicating that the two phenotypes coexist in HD-hCOs, and some that diverged from the progenitor identity were retained in a stagnant stage before differentiating into mature neurons. In HD-hCOs, a population of cells that do not express SOX2 or NEUN strongly supported the incomplete or delayed maturation of these progenitors.

Based on the exceptional accumulation of CTIP2+ cells and the dysregulated fate specification of specific cortical neuron subtypes in the HD group, we used CTIP2 antibody immunostaining, a specific neuronal layer marker, to characterize neurons with delayed maturation (Fig. 3A–D, S7B). The co-immunostaining of SOX2, NEUN, and CTIP2 antibodies showed that CTIP2+ cells nested around the VZ-like zones, and the expression levels of CTIP2 in neurons showed a gradient in the CTR group (Fig. 3D), similar to human fetal brain staining (Fig. S5A). CTIP2+ cells were scattered around the VZ-like zones, and the expression levels of CTIP2 in most cells were lower in the HD group than in the CTR group (Fig. 3B, C). In the chimeras, the fluorescence intensity of HD CTIP2+ cells was significantly lower than that of CTR cells in the CP region of the same neural tube (Fig. S8A, S8B).

Fig. 3: Delayed maturation of postmitotic neurons in HD hCOs.

A Representative images of immunostaining with SOX2, CTIP2 and NEUN antibodies in HD and CTR hCOs on Day 60. The left panel is CTIP2+ cells in hCOs (displayed as gradient grey value). B, C Representative images (B) showing the distribution tendency of CTIP2+SOX2-NEUN+ / CTIP2+SOX2+NEUN-/CTIP2+SOX2-NEUN- cells in the different layers of HD and CTR hCOs. Pie charts (C) showing the percentages of each type on CTIP2+ cells (n = 6) on Day 60 and 80. Data, mean ± s.e.m. D The graph of the intensity (n = 6) of CTIP2 antibody staining in three types of cells (CAG 55 and 59 vs. H9 and CAG 19) as described in C. Data, mean ± s.e.m. One-way ANOVA. *P < 0.05; ns, nonsignificant. E Immunostaining of NEUN, CTIP2 and DCX antibodies in HD and CTR hCOs on Day 60 (yellow arrows, typical DCX+ cells in VZ-like area). Comparing the CTIP2 immunostaining intensity (n = 8) of DCX/ NEUN in HD with CTR (CAG 55 and 59 vs. H9 and CAG 19). Data, mean ± s.e.m. One-way ANOVA. *P < 0.05; ns, nonsignificant. F The method of measuring DCX+ cell angle and the typical image for analysis. Scatter plots show the tendency of angle (n = 8) on Day 60 CTR/HD hCOs (CAG 55 and 59 vs. H9 and CAG 19). G Representative images of CTR/HD hCOs (CAG 55 and 59 vs. H9) stained with Ca2+ indicators for Ca2+ imaging on Day 150 (left panel). The heatmap showed the calcium traces normalized by the min-max (middle panel). Representative images indicate the calcium activity traces of individual neurons in CTR/HD hCOs (right panel). The red dots represent peaks, and the green represents bases. H. Quantifications of the average amplitude of ΔF/F per cell and calcium spike frequency of neurons from CTR/HD hCOs. Data, t-test. **P < 0.01.

In the CTR group, CTIP2+ cells highly overlapped with NEUN+ cells, whereas they were mutually exclusive within the SOX2+ regions (Fig. 3C). In contrast, all three markers displayed a scattered pattern in the HD group, and we identified many cells expressing CTIP2 but not other markers (Fig. 3C). These separate CTIP2+ cells appeared to lose the progenitor fate and were strangled at the intermediate stage. To track the destination of CTIP2+ cells in HD-hCOs, we identified CTIP2+ cells in both the CTR and HD groups. In hCOs from days 60 to 80, we found that there were three subpopulations of CTIP2+ cells: CTIP2+SOX2-NEUN+ cells represented mature CTIP2+ neurons; CTIP2+SOX2+NEUN- cells included CTIP2+ cells with progenitor fate; and CTIP2+SOX2-NEUN- cells. Only CTIP2+ cells were at an intermediate stage between the progenitor and mature cells (Fig. 3C, S7C). Generally, on days 60 and 80, significant CTIP2+ cell populations were CTIP2+SOX2-NEUN+ cells in the CTR group and CTIP2+SOX2-NEUN- cells in the HD group (Fig. 3C). We also observed that CTIP2 and SOX2 expression were not mutually exclusive (Fig. 3B, C). CTIP2+SOX2+NEUN- cells could be identified in both the CTR and HD groups. The percentage of CTIP2+SOX2+NEUN- cells consistently decreased from days 60 to 80, revealing the equal potential of CTIP2+SOX2+ cells to leave the progenitor fate in both groups (Fig. 3E). The percentage of CTIP2+SOX2-NEUN+ cells in the CTR group increased from days 60 to 80 (Fig. 3C). However, the percentage of CTIP2+SOX2-NEUN+ cells decreased in the HD group, and the percentage of CTIP2+SOX2-NEUN- cells increased significantly (Fig. 3C). Our results suggest that the exceptional accumulation of CTIP2+ cells in HD-hCOs may be due to delayed maturation. Moreover, all three HD group populations had the same levels of CTIP2 expression (Fig. 3D), indicating that CTIP2 expression is a consequence, but not the primary inducer, of delayed neural maturation.

To define the delayed maturation of CTIP2+ cells in HD-hCOs, we used doublecortin (DCX) antibody staining, a marker of immature and post-mitotic migrating neurons, to characterize their fate (Fig. 3E). Most DCX+ cells spanned the entire cortical plate, and a few were nested in the VZ-like zones (Fig. 3E). Almost all CTIP2+ cells expressed DCX, including NEUN- cells in the HD group. The DCX expression level in CTIP2+ cells in the HD group was slightly higher than that in the CTR group; however, the expression level of NEUN decreased (Fig. 3E). Migrating DCX+ cells were present in the VZ-like zones of hCOs (Fig. 3E, F). To further investigate the possible cause of delayed maturation in DCX+ neurons, we quantified the migrating DCX+ cells by analyzing their positions in the substratum plane (Fig. 3F). In the VZ-like zone of the CTR group, the scattered DCX+CTIP2+NEUN+ cells were oriented perpendicular to the substratum plane (the angle between the cell axis and the assumed apical surface≈90°), and most migrating DCX+ neurons were 20 μm away from the apical surface, showing robust radial migration (Fig. 3F). In contrast, DCX+CTIP2+ scattered cells did not express NEUN in the VZ-like zone of HD-hCOs, corresponding to the location of DCX+CTIP2+NEUN+ cells in the CTR group (Fig. 3E). Meanwhile, the CTIP2+DCX+NEUN- cell angle to the apical surface was more variable, and migrating neurons were within 20 μm from the assumed apical surface, similar to tumbling in place (Fig. 3F). Our analyses suggest that the delayed maturation of postmitotic neurons in HD- hCOs may result in the retention of CTIP2+ cells in the CP-like region, causing dysregulated fate specification of specific cortical neuron subtypes. To see the characteristics of cortical neuronal network in HD-hCOs, we performed calcium imaging with the fluorescence dyes (Cal-520 indicator) [45] in CTR/HD-hCOs cultured for 150 days and found that the frequency and the average amplitude of calcium activities in the neurons from HD-hCOs were significantly lower compared with those from CTR group (Fig. 3G, H). This result suggested that the altered corticogenesis in HD-hCO disrupts the cortical neuronal network of HD-hCOs.

HD cortical projections aberrantly target striatal organoids early

Subcortical projections were constructed using an assembloid model to determine whether delayed maturation disrupts subcortical projections. The human cerebral cortex establishes projections toward the striatum [46]. We fused CTR or HD-hCOs tagged with GFP and healthy human striatal organoids (hStrOs) to create hC-Stro assembloids (Fig. 4A). Upon fusion of hCO with hStrOs for 30 days, we observed projection-like fibers in hCOHD-hStrO that aggregated into bundles protruding from the fluorescent margin of HD-hCOs and merged into hStrO after 5 days after fusion (daf) (Fig. S8C). Notably, the GFP+ positive spindle originated from the margin of the proximal fusion site, traversed the striatum, reached the distal part, and expanded as fusion proceeded (Fig. 4B, C). However, we did not observe any apparent projection/spindle protruding from a cortical organoid derived from the CTR group during the monitoring period (Fig. 4C). We further sectioned and stained the fused organoids to characterize the subcortical projections on 12, 20, and 30 daf. Based on the method of measuring the fused organoid [47], we calibrated the area of the GFP+ mass in a normalized region (see Methods) (Figs. 4D, E, S9A). The data showed that the volume of the GFP+ region (S R1/2/3) in hCOHD -hStro was higher than that in hCOCTR-hStro at all three time points (Fig. 4E). Most GFP+MAP2+ neurons expressed the vesicular glutamate transporter (vGLUT1), indicating their glutamatergic identity in the assembloids (Fig. S9B).

Fig. 4: Formation of early projections from HD hCO in hC-StrO assembloids.

A, B Schematic (A) of generating hC-StrO assembloids using GFP+ hCO and GFP− hStrO and representative images (B) of hC-Stro assembloids. C Epifluorescence microscopy images showed the subcortical projections protruding from GFP+ hCO in hC-Stro assembloids (yellow arrows, GFP expressing projections). D Immunostaining revealed GFP-expressing projections in hC-StrO assembloids on Day 12, 20, and 30 (daf). The projections were immunostained by a GFP antibody. E The method of measuring GFP-expressing targeting projections in hC-Stro assembloids and comparing GFP+ projections area of HD and CTR hC-StrO assemblies on Day 12, 20, and 30 (daf). Data, mean ± SD. One-way ANOVA. *P < 0.05; **P < 0.01; ns, nonsignificant. F Immunostaining with Bassoon and PSD95 antibodies revealed the synapses on GFP-expressing projections in hC-StrO assembloids. Synapses are identified by the colocalization of presynaptic (red; Bassoon) with postsynaptic markers (green, PSD95). Neural surfaces were rendered, and PSD95+ puncta in a neuron were counted using Imaris 9.7 software (the top left panel). G Comparing the synapses on GFP expressing projections of HD with CTR of hC-StrO assembloids. The overlapped signal of Bassoon with PSD95 is defined as a synapse. Data, means ± s.e.m. One-way ANOVA. *P < 0.05; ns, nonsignificant.

Synapse formation is the hallmark of neuronal maturation [48]. To determine whether the projections of HD-hCOs were mature, we used Bassoon, a presynaptic marker, and PSD95, a postsynaptic marker, to stain GFP+ projections across striatal organoids. The Bassoon+ puncta paralleled the PSD95+ puncta on projections in 3D rendered images (Fig. 4F, G), and the synaptic puncta count on HD-hCO projections was higher than that on CTR-hCO projections on 12 and 20 daf and reached the same level on 30 daf (Fig. 4G). These data suggest that HD-hCOs form mature subcortical projections earlier than CTR-hCOs.

PolyQ assemblies with mHTT lead to deficient Golgi apparatus, clathrin+ vesicles and the