Dear Editor,
Although brain development and expansion critically depend on neurogenesis and gliogenesis, the mechanisms driving cerebral cortical gyrification and enlargement in primates remain incompletely understood (
Akula et al., 2023). Among the many genes linked to microcephaly primary hereditary (MCPH), mutations in the abnormal spindle-like microcephaly-associated (
ASPM) gene are the most common cause (
Bond et al., 2002;
Létard et al., 2018).
ASPM is predominantly expressed in the progenitor cells of the developing brain, specifically in the ventricular and subventricular zones of the cerebral cortex, where its expression peaks during neurogenesis and declines postnatally (
Bond et al., 2002). This developmentally regulated expression underscores its critical role in the expansion of the cerebral cortex. However, the pathological consequences of
ASPM mutation in non-human primates remain unexplored. Given the lack of cerebral cortical gyrification in rodents as well as other small animals and the evolutionarily divergent functions of
ASPM (
Montgomery and Mundy, 2014), it is important to use larger animal models that exhibit gyrification to investigate
ASPM function.
We used CRISPR/Cas9 to target exon 3 and exon 9 of the monkey ASPM gene to generate a complete knockout (Fig. 1A; Table S1). ASPM gRNA and Cas9 mRNA were injected into fertilized cynomolgus monkey eggs, which were then transferred to surrogate females, resulting in six live births (Fig. 1B). Using placental tissue from newborns or brain tissue from abortions, we verified ASPM editing by using a T7E1 assay for exon 3 and a BsmAI digest assay for exon 9, as the exon 9 target sequence contains a BsmAI recognition site (Fig. S1A). Of the six newborns, one (ASPM KO, male) displayed an obviously smaller head and body compared to an age-matched male control monkey born on the same day (Fig. 1C). The other five newborns showed normal development and activity. Analysis of blood samples indicated these monkeys retained a wild-type ASPM allele. Their lack of an abnormal phenotype suggests the persistence of functional ASPM in the brain, consistent with a heterozygous or mosaic state. For this reason, we concentrated further investigation on the ASPM KO monkey, which is putatively homozygous for the mutation, and its genetically matched control.
The ASPM KO monkey consistently exhibited a smaller body and head at 3, 6, and 9 months of age (Fig. S1B). This mutant also showed reduced movement and exploratory activity compared to the control (Fig. S1C and Video S1). The ASPM KO monkey unfortunately died from pneumonia at 9 months of age, which prevented the longitudinal analysis of its behavioral phenotypes. Analysis of peripheral tissues from the deceased ASPM KO monkey revealed pulmonary edema, with no significant morphological changes in the liver, heart, or kidney (Fig. S2). Measurements of head circumference, body weight, and brain weight revealed that the whole brain weight of the ASPM KO monkey was 28.4% of the control, while its body weight was 62.8% of the control (Fig. 1D and 1E). Sectioning of the prefrontal cortex clearly showed a much smaller cortex in the ASPM KO monkey compared to the control (Fig. 1F). Immunocytochemistry of the prefrontal cortex also confirmed the elimination of ASPM protein expression in the KO monkey (Fig. 1G). Using PCR with primers specific for the targeted sequences in exons 3 and 9, we found that exon 3 was completely disrupted while exon 9 was partially mutated in different tissues of the ASPM KO monkey (Fig. 1H). DNA sequencing revealed a 9 bp deletion in exon 9 and three distinct frameshift mutations in exon 3—a 2 bp insertion, a 5 bp deletion, and a 246 bp deletion—all predicted to cause premature termination of the ASPM protein (Fig. 1I).
Although the cortical gray matter was much thinner in the
ASPM KO monkey (Figs. 2A and S31), the six cortical layers remained intact (Fig. 2B). However, in the thinner cortical layers of the
ASPM KO monkey, the density of NeuN-positive cells appeared increased, and fewer cells exhibited long neurites compared to the control (Fig. 2C). Western blot of ASPM KO brain tissue showed that DCX, a marker of newborn neurons, was elevated (Fig. 2D), suggesting that
ASPM KO neurons are less mature. Single-cell RNA-seq analysis of the non-human primate brains indicated that
ASPM is expressed in oligodendrocytes (
Han et al., 2022; Fig. S3B). Notably, oligodendrocyte density was remarkably reduced in the
ASPM KO monkey (Fig. 2E), while astrocytes and microglia were unaltered (Fig. S4). Quantification confirmed a decrease in oligodendrocyte density and an increase in neuronal density (Fig. 2F). Consistent with the immunocytochemical staining, myelin proteins (MBP, Olig2, and MOG), which are produced by oligodendrocytes, were also decreased, as shown by Western blot (Fig. 2G) and quantification of their ratios to vinculin (Fig. 2H). In accordance with the important role of myelination in maintaining neuronal synapses (
Wang et al., 2018), Western blot also demonstrated reductions in synaptic proteins (PSD95, synaptophysin, SNAP25, and MAP2) in the
ASPM KO monkey (Fig. 2I).
While
Aspm KO mice display only mild microcephaly (4%–12% reduction) (
Fujimori et al., 2014;
Jayaraman et al., 2016;
Pulvers et al., 2010;
Williams et al., 2015) and humans show a > 50% reduction in cortical volume (
Passemard et al. 2016), the
ASPM KO monkey exhibits a remarkably severe phenotype with over 70% reduction in brain weight. This finding underscores the critical importance of large animal models with a gyrated cerebral cortex for modeling
ASPM function. The phenotype was also more severe than in the
Aspm KO ferret, which shows a 20%–40% decrease in brain weight (
Johnson et al., 2018). We compared
ASPM mutations and associated phenotypes across animal models.
ASPM undergoes alternative splicing, resulting in different isoforms in humans and mice (Fig. S5). Human
ASPM has two additional truncated isoforms that lack exons 4–17 and are absent in mice (Fig. S5). In the
Aspm KO ferret model, TALEN-mediated targeting of exon 15 created a single exon mutation that also occurs in humans (
Johnson et al., 2018). While patient-derived brain organoids with
ASPM mutations (exons 18 and 25) revealed a neurogenesis defect (
Li et al., 2017), examining oligodendrocyte-related phenotypes in organoids remains challenging. In our
ASPM KO monkeys, however, two exons (3 and 9) were targeted. Targeting both exon 3 and exon 9 is likely to eliminate the expression of all protein isoforms and may therefore result in loss of oligodendrocytes in the developing brain and more severe microcephaly.
The complete loss of
ASPM function, resulting from multi-exon targeting, may underlie the selective reduction of oligodendrocytes—a phenotype not reported in other
ASPM KO animals. Gliogenesis in the outer subventricular zone, a brain region absent in rodents, plays a critical role in the expansion and gyrification of the primate cerebral cortex (
Rash et al., 2019). Recent studies of human postnatal cortical development have revealed that myelination, a key function of oligodendrocytes, is critical for infant cortical development and correlates with cortical thickness (
Natu et al., 2019). A striking observation in our study was the severe deficit of oligodendrocytes in the
ASPM KO monkey, which was not accompanied by significant abnormalities in astrocytes or microglia. This selective loss suggests that
ASPM plays a critical and specific role in the generation of oligodendrocytes during early brain development. As a centrosomal protein essential for mitotic spindle formation, the loss of
ASPM likely disrupts the division and proliferation of oligodendrocyte progenitor cells. Since oligodendrocyte generation is critical for cortical expansion in primates, this specific deficit could explain the more severe microcephaly observed in
ASPM-knockout primates compared to rodents.
A limitation of our study is that only one ASPM KO monkey was successfully generated among six live births, which limits the statistical power and generalizability of the findings. The rarity of biallelic knockout animals in non-human primates (NHPs) is a common challenge due to the inefficiencies of CRISPR/Cas9 editing and ethical constraints. Furthermore, the ASPM KO monkey died at 9 months of age from pneumonia, preventing long-term observation of developmental trajectories, behavioral outcomes, or potential compensatory mechanisms. Despite these limitations, our study provides compelling evidence for ASPM’s role in primate brain development and highlights the value of NHP models for studying neurodevelopmental disorders. Future research should explore the molecular pathways through which ASPM regulates oligodendrocyte development and investigate potential therapeutic strategies to mitigate the effects of ASPM deficiency.
The Author(s) 2025. Published by Oxford University Press on behalf of Higher Education Press.
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