Entosis implicates a new role for P53 in microcephaly pathogenesis, beyond apoptosis.

Entosis, a form of cell cannibalism, is a newly discovered pathogenic mechanism leading to the development of small brains, termed microcephaly, in which P53 activation was found to play a major role. Microcephaly with entosis, found in Pals1 mutant mice, displays P53 activation that promotes entosis and apoptotic cell death. This previously unappreciated pathogenic mechanism represents a novel cellular dynamic in dividing cortical progenitors which is responsible for cell loss. To date, various recent models of microcephaly have bolstered the importance of P53 activation in cell death leading to microcephaly. P53 activation caused by mitotic delay or DNA damage manifests apoptotic cell death which can be suppressed by P53 removal in these animal models. Such genetic studies attest P53 activation as quality control meant to eliminate genomically unfit cells with minimal involvement in the actual function of microcephaly associated genes. In this review, we summarize the known role of P53 activation in a variety of microcephaly models and introduce a novel mechanism wherein entotic cell cannibalism in neural progenitors is triggered by P53 activation.

P53 independent pathogenic mechanisms contribute to BubR1 microcephaly.

The mosaic variegated aneuploidy (MVA)-associated gene Budding Uninhibited by Benzimidazole 1B (BUB1B) encodes BUBR1, a core member of the spindle assembly checkpoint complex that ensures kinetochore-spindle attachment for faithful chromosome segregation. BUB1B mutation in humans and its deletion in mice cause microcephaly. In the absence of BubR1 in mice, massive cell death reduces cortical cells during neurogenesis. However, the molecular and cellular mechanisms triggering cell death are unknown. In this study, we performed three-dimensional imaging analysis of mitotic BubR1-deficient neural progenitors in a murine model to show profound chromosomal segregation defects and structural abnormalities. Chromosomal defects and accompanying DNA damage result in P53 activation and apoptotic cell death in BubR1 mutants. To test whether the P53 cell death pathway is responsible for cortical cell loss, we co-deleted Trp53 in BubR1-deficient cortices. Remarkably, we discovered that residual apoptotic cell death remains in double mutants lacking P53, suggesting P53-independent apoptosis. Furthermore, the minimal rescue of cortical size and cortical neuron numbers in double mutant mice suggests the compelling extent of alternative death mechanisms in the absence of P53. This study demonstrates a potential pathogenic mechanism for microcephaly in MVA patients and uncovers the existence of powerful means of eliminating unfit cells even when the P53 death pathway is disabled.

An entosis-like process induces mitotic disruption in Pals1 microcephaly pathogenesis.

Entosis is cell cannibalism utilized by tumor cells to engulf live neighboring cells for pro- or anti-tumorigenic purposes. It is unknown whether this extraordinary cellular event can be pathogenic in other diseases such as microcephaly, a condition characterized by a smaller than normal brain at birth. We find that mice mutant for the human microcephaly-causing gene Pals1, which exhibit diminished cortices due to massive cell death, also exhibit nuclei enveloped by plasma membranes inside of dividing cells. These cell-in-cell (CIC) structures represent a dynamic process accompanied by lengthened mitosis and cytokinesis abnormalities. As shown in tumor cells, ROCK inhibition completely abrogates CIC structures and restores the normal length of mitosis. Moreover, genetic elimination of Trp53 produces a remarkable rescue of cortical size along with substantial reductions of CIC structures and cell death. These results provide a novel pathogenic mechanism by which microcephaly is produced through entotic cell cannibalism.

Nearly complete deletion of BubR1 causes microcephaly through shortened mitosis and massive cell death.

BUB-related 1 (BubR1) encoded by Budding Uninhibited by Benzimidazole 1B (BUB1B) is a crucial mitotic checkpoint protein ensuring proper segregation of chromosomes during mitosis. Mutations of BUB1B are responsible for mosaic variegated aneuploidy (MVA), a human congenital disorder characterized by extensive abnormalities in chromosome number. Although microcephaly is a prominent feature of MVA carrying the BUB1B mutation, how BubR1 deficiency disturbs neural progenitor proliferation and neuronal output and leads to microcephaly is unknown. Here we show that conditional loss of BubR1 in mouse cerebral cortex recapitulates microcephaly. BubR1-deficient cortex includes a strikingly reduced number of late-born, but not of early-born, neurons, although BubR1 expression is substantially reduced from an early stage. Importantly, absence of BubR1 decreases the proportion of neural progenitors in mitosis, specifically in metaphase, suggesting shortened mitosis owing to premature chromosome segregation. In the BubR1 mutant, massive apoptotic cell death, which is likely due to the compromised genomic integrity that results from aberrant mitosis, depletes progenitors and neurons during neurogenesis. There is no apparent alteration in centrosome number, spindle formation or primary cilia, suggesting that the major effect of BubR1 deficiency on neural progenitors is to impair the mitotic checkpoint. This finding highlights the importance of the mitotic checkpoint in the pathogenesis of microcephaly. Furthermore, the ependymal cell layer does not form in the conditional knockout, revealing an unrecognized role of BubR1 in assuring the integrity of the ventricular system, which may account for the presence of hydrocephalus in some patients.

Retrogradely Transportable Lentivirus Tracers for Mapping Spinal Cord Locomotor Circuits.

Retrograde tracing is a key facet of neuroanatomical studies involving long distance projection neurons. Previous groups have utilized a variety of tools ranging from classical chemical tracers to newer methods employing viruses for gene delivery. Here, we highlight the usage of a lentivirus that permits highly efficient retrograde transport (HiRet) from synaptic terminals within the cervical and lumbar enlargements of the spinal cord. By injecting HiRet, we can clearly identify supraspinal and propriospinal circuits innervating motor neuron pools relating to forelimb and hindlimb function. We observed robust labeling of propriospinal neurons, including high fidelity details of dendritic arbors and axon terminals seldom seen with chemical tracers. In addition, we examine changes in interneuronal circuits occurring after a thoracic contusion, highlighting populations that potentially contribute to spontaneous behavioral recovery in this lesion model. Our study demonstrates that the HiRet lentivirus is a unique tool for examining neuronal circuitry within the brain and spinal cord.

Effects of αTAT1 and HDAC5 on axonal regeneration in adult neurons.

The role of posttranslational modifications in axonal injury and regeneration has been widely studied but there has been little consensus over the mechanism by which each modification affects adult axonal growth. Acetylation is known to play an important role in a variety of neuronal functions and its homeostasis is controlled by two enzyme families: the Histone Deacetylases (HDACs) and Histone Acetyl Transferases (HATs). Recent studies show that HDAC5 deacetylates microtubules in the axonal cytoplasm as part of an injury-induced regeneration response, but little is known about how acetylation of microtubules plays a role. Alpha-tubulin acetyl transferase (αTAT1) is a microtubule specific acetyl transferase that binds to microtubules and directly affects microtubule stability in cells. We hypothesize that increasing tubulin acetylation may play an important role in increasing the rate of axonal growth. In this study, we infected cultured adult DRG neurons with αTAT1 and αTAT1-D157N, a catalytically inactive mutant, and HDAC5, using lentiviruses. We found that αTAT1 significantly increases tubulin acetylation in 293T cells and DRG neurons but αTAT1-D157N does not. Furthermore, in neurons infected with αTAT1, a significant increase in acetylated tubulin was detected towards the distal portion of the axon but this increase was not detected in neurons infected with αTAT1-D157N. However, we found a significant increase in axon lengths of DRG neurons after αTAT1 and αTAT1-D157N infection, but no effect on axon lengths after infection with HDAC5. Our results suggest that while αTAT1 may play a role in axon growth in vitro, the increase is not directly due to acetylation of axonal microtubules. Our results also show that HDAC5 overexpression in the axonal cytoplasm does not play a crucial role in axonal regeneration of cultured DRG neurons. We expressed these genes in DRG neurons in adult rats and performed a sciatic nerve crush. We found that axons did not regenerate any better when infected with any of the constructs compared with control animals. Thus, while αTAT1 may be important for axonal growth in vitro, neither αTAT1 nor HDAC5 had an effect in vivo on the regeneration of sciatic nerves.