When Building Goes Wrong: Microcephaly, Zika and Lissencephaly

Two children can both have a malformed cerebral cortex for opposite reasons. In one, the cortex is small, but its layers are in their usual order. In the other, the cortex is thick and its surface nearly smooth, and many of its neurons sit in the wrong place.

The first child has primary microcephaly. The second has lissencephaly, a word that means “smooth brain”. Each points to one step of building a cortex that failed, and to genes needed for that step. The Zika epidemic of 2015 and 2016 showed that an infection can strike the same step as a gene.

The idea: a cortex can go wrong in number or in place

Recall how the cortex is built. Radial glia, the progenitor cells that line the fluid-filled ventricle, divide to make more of themselves and then to make neurons (that lesson). Each newborn neuron then moves outward along a radial glial fiber to its layer, later-born neurons passing the earlier ones (that lesson). Two jobs, so two kinds of failure:

• Too few neurons made. Progenitors stop dividing early or die, and the cortex is small, though its plan can look normal. This is microcephaly.
• Neurons made but misplaced. Neurons stall on the way to their layer. This gives lissencephaly, or a heterotopia: a cluster of neurons sitting where they do not belong.

Microcephaly is defined at the bedside, not under a microscope. It means a head circumference, measured around the skull just above the eyebrows and ears, well below the average for the child’s age and sex. The World Health Organization uses a cutoff of more than two standard deviations below the mean, and calls more than three severe. A small head is a sign, not a diagnosis, with many possible causes. This lesson follows one group of genes and one virus, which act on the same cells.

For each malformation, ask the question you would ask of any mouse mutant: which step failed?

Too few: genes at the spindle pole

Primary microcephaly (MCPH) is present at birth and inherited as a recessive trait: a child is affected when both copies of a gene are broken. That is why it is found most often in families in which the parents are related. The brain is small, and the cerebral cortex is reduced most. Its layers are broadly normal, though its folds are often simpler. Intellectual disability is common and varies in degree.

More than twenty MCPH genes are now known. In many studied populations the commonest cause is ASPM, which Geoffrey Woods and colleagues linked to the disease in 2002. The name records its origin: abnormal spindle-like, microcephaly associated, after the fly gene abnormal spindle. Others include MCPH1 (microcephalin), CDK5RAP2 and WDR62. Most of their proteins sit at the centrosome or the spindle pole. The centrosome is the cell’s main microtubule-organizing center. At mitosis the two centrosomes form the poles of the spindle that pulls the chromosomes apart.

Why would a spindle protein matter more for the brain than for other organs? A leading idea is about timing. Early in development, progenitors divide symmetrically, each division making two progenitors (that lesson). Later they switch to divisions that make neurons. Each round of symmetric division doubles the pool:

progenitors at the switch = starting progenitors × 2^n, where n is the number of symmetric rounds

In this simple model, switching one round early halves the pool, and with it the number of neurons the pool will make. A defect in how the spindle is placed, or in how long mitosis takes, could tip a progenitor toward the switch, or toward death. In mouse embryos, knocking down Aspm tilted the plane of progenitor division and shifted divisions away from the symmetric, progenitor-making kind (Fish and colleagues, 2006).

There is a puzzle. A child with no working ASPM has a markedly small brain, but mice lacking Aspm have only a mild reduction. In ferrets, whose cortex is folded and rich in outer radial glia, an Aspm knockout reduced the cortex much more (Johnson and colleagues, 2018). One reading is that a cortex that grows through more rounds of division, and through more kinds of progenitor, is more exposed to a small defect in each division. That remains a hypothesis; that lesson takes up how the human cortex got large.

Zika: a virus that finds the progenitors

Zika is a flavivirus, a relative of dengue, spread mainly by Aedes mosquitoes. It was first isolated in 1947, from a monkey in the Zika Forest of Uganda, and for decades it was known for mild illness. In 2015 it spread through Brazil. Late that year, doctors in the country’s northeast reported many more newborns with microcephaly than usual. Brazil declared a national public health emergency in November 2015, and the World Health Organization declared a Public Health Emergency of International Concern on 1 February 2016.

A rise in cases alongside a virus does not prove cause. The case was built from three kinds of evidence pointing the same way.

1. Epidemiology. The rise in microcephaly followed the virus in time and place. A look back at a 2013 to 2014 outbreak in French Polynesia found an increase in microcephaly there too, with timing that fit infection in the first trimester.
2. Pathology. In 2016 Mlakar and colleagues reported Zika virus RNA and virus particles in the brain of a fetus whose mother had been infected in Brazil during pregnancy. The brain was small, with calcifications.
3. Laboratory models. In March 2016 the laboratories of Guo-li Ming, Hongjun Song and Hengli Tang reported that Zika infects human cortical neural progenitors grown from stem cells far more readily than it infects the stem cells or immature neurons, that it kills some of the progenitors and slows the growth of the rest. Stevens Rehen’s group in Rio de Janeiro reported that the virus stunts human neurospheres and brain organoids, tissue grown in three dimensions from stem cells (that lesson).

In April 2016 Sonja Rasmussen and colleagues at the US Centers for Disease Control and Prevention weighed this evidence against formal criteria for calling an agent a cause of birth defects. They concluded that Zika virus causes microcephaly and other serious brain defects. The pattern in affected infants, now called congenital Zika syndrome, includes severe microcephaly, calcium deposits in the brain, damage to the eye and stiff joints.

Two cautions. Much of the mechanism comes from cultured cells, organoids and mice, so it shows what the virus can do to human progenitors, not what it does in each fetus. And most infections in pregnancy do not produce microcephaly; the risk appears highest when infection comes early. Still, it fits the first category: the virus attacks the cells that make neurons, so the cortex is short of neurons from the start.

In the wrong place: lissencephaly and the double cortex

“Human lissencephaly” is the other category. A normal human cortex is folded and has six layers. In classic lissencephaly it is thick, has few or no folds, and is organized into about four crude layers. Affected children usually have severe intellectual disability and epilepsy. The progenitors did their job; the neurons failed to travel.

Among “the mutants” of migration (LIS1, NudE and Ndel1, doublecortin, CDK5, dynein and more), two were found through children.

LIS1. In 1993 Orly Reiner, David Ledbetter and colleagues isolated a gene from chromosome region 17p13.3, which is deleted in Miller-Dieker syndrome, a severe lissencephaly with distinctive facial features. They named it LIS1. The deletion removes one copy of LIS1, and mutations of LIS1 alone cause isolated lissencephaly, though the other copy is normal. Half the usual amount of LIS1 is not enough, a situation called haploinsufficiency. The larger Miller-Dieker deletion also removes neighbouring genes, including the one for 14-3-3ε, which is thought to add to the severity.

The sequence did not explain the disease. A clue came in 1995 from a mould, Aspergillus nidulans, which needs a gene called nudF to spread its nuclei along its growing filaments. NudF proved to be 42 percent identical to human LIS1, and other nud genes of the same mould encode parts of cytoplasmic dynein, a motor that walks toward the minus ends of microtubules. In mammalian cells LIS1 binds dynein and the NudE-family proteins NDE1 and NDEL1. LIS1 is needed to move the nucleus: when LIS1 was knocked down in rat embryos (Tsai and colleagues, 2005), migrating neurons kept extending their leading process, but the cell body and nucleus barely moved. Progenitor nuclei also stopped their up-and-down movement in the ventricular zone. The mechanics are in that lesson; the logic is that a nucleus that cannot be moved leaves its neuron short of its layer.

Doublecortin. Some families showed a striking pattern. Affected boys had lissencephaly, while their mothers or sisters had a milder disorder, subcortical band heterotopia, or “double cortex”: a band of gray matter lying in the white matter beneath a nearly normal cortex. That pointed to a gene on the X chromosome. In 1998 two groups reported it in the same issue of Cell: Joseph Gleeson, Christopher Walsh and colleagues in Boston, and Vincent des Portes, Jamel Chelly and colleagues in Paris. The gene, DCX, encodes doublecortin, a protein that binds microtubules and stabilizes them in migrating neurons.

Why the sex difference? A male has one X. If it carries the mutation, no neuron makes working doublecortin, and most neurons stall: lissencephaly. A female has two X chromosomes, and early in development each cell silences one of them at random, a process called X inactivation. Her cortex is a mosaic. On the standard interpretation, neurons with the normal X active reach the cortex. Neurons with the mutant X active stall partway and form the band. The usual reading is that each neuron follows its own active X, so doublecortin acts within the migrating cell.

The two genes also leave different maps. Lissencephaly from LIS1 is usually worse toward the back of the brain, and from DCX toward the front. A third gene links to an earlier lesson: mutations in both copies of RELN, the human gene for Reelin, cause lissencephaly with a small cerebellum, a human counterpart of the reeler mouse (that lesson).

Design problem

You study two mouse lines. In the first, cortical progenitors switch early from symmetric proliferative divisions to neuron-producing divisions. In the second, neurons are born on schedule but cannot move their nuclei along radial glia. Predict the brain size and the layering of the cortex in each line, and say which human condition each resembles.