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Your Brain

Smoother Path to Understanding Smooth Brains

Introduction

The LIS1 gene, infamous due to its removal having the capability to cause lissencephaly – meaning "smooth brain" in Latin; was first identified in patients suffering from seizures and these were all found to be caused by deletions and/or mutations in the 17p13.3 region. This gene was studied exhaustively due to its clinical relevance for over 20 year. When it comes to
brain development, LIS1 is one of the first genes which come to mind, due to the fact that it has been known for a long time that hemizygote mutations in the gene caused the severe brain malformation, lissencephaly.

Lissencephaly is mostly associated with the Miller-Dieker syndrome – a developmental disorder that affects cortical neuron migration, giving the brain surface a smooth appearance. LIS1 is a subunit of platelet-activating-factor acetyl hydrolase (PAFAH1B1), but a critical one and with plenty of functions. Previous studies have shown that even loss of one allele of the LIS1 gene causes lissencephaly (Reiner et al, 1993) because haploinsufficiency of the LIS1 gene is enough to cause it. Furthermore reduction of LIS1 expression also has been shown to cause neuronal migration defects (Hirotsune et al, 1998) apart from the phenotypes of lissencephaly, thus indicating high levels of sensitivity to dosage of the LIS1 product. To make matters worse, recent findings now show that increased LIS1 dosage also causes defects in human and mouse brain development (Bi et al., 2009).

LIS1 gene was first made famous in the 1980's when it was identified as the main causer of lissencephaly; and since it was the first gene to be identified which was associated with this disorder, hence the number 1 was used to make the name LIS1. Lissencephaly is found once in every 30000 births and is a condition where the brain lacks a high proportion of the convolutions of the brain. Ones who are born with lissencephaly suffer from recurrent seizures, have abnormal cortical organisation and do not reach normal developmental stages. The dosage of the LIS1 gene product seems to be critical since it has been shown many times (Reiner et al., 1993; Lo Nigro et al., 1997; Fogli et al., 1999) that even deletions or point mutations in one allele of the gene is enough for brain malformation. Even though the LIS1 gene has been around for over 20 years, understanding how and why LIS1 causes brain malformation and the many other disruptions still requires further study. What is known however, since the LIS1 gene belongs to the WD repeat protein family it is expected to be involved in many protein-protein interactions and this seems to be case also for the LIS1 gene. In fact, LIS1 interacts with a large number of proteins (reviewed by Reiner, 2000) including doublecortin, dynein and dynactin which are all microtubule associated proteins (of all sorts, including the mitotic spindle). Early studies were carried out in familiar fashion, which is by partial deletion of the gene resulting in a truncated and/or inactive protein; then looking at what went wrong in the organism. When the first exon of the LIS1 gene was deleted, a shorter LIS1 protein was formed which caused homozygotes to be unviable, thus no information could be gained from them. However, heterozygotes were viable and also fertile (Cahana et al., 2001) thus all research was done in these mutants. This way many functions of the LIS1 protein were identified and first one to be observed was that this protein was simply not working on its own but as a subunit of the platelet-activating-factor-acetyl-hydrolase (PAFAH); thus the formal name of the LIS1 gene was changed to PAFAH1B1.
Although there were suggestions of a possible role of LIS1 in mitosis (Faulkner et al., 2000) before Cahana and his co-workers published their findings in mice, evidence from these studies showed LIS1 does play a role in mitosis because the mitotic spindles in LIS1
overexpressing cell were oriented randomly.

In humans trying to find the role of LIS1 has been tricky and every so often in vain as genetic rearrangement mutations are quite common in humans and usually affect multiple genes simultaneously. This is the case with individuals who either have a deletion or a duplication
at the 17p13.3 region – where LIS1 is located. PAFAH1B1 is only a mega base away from a multiple of genes including the YWHAE gene which was also shown to cause severe brain malformations and has a role in pathways LIS1 is also thought to have a role in (Pramparo et al., 2011). The 17p13.3 region is often called the MDS region (MDS: Miller-Dieker syndrome) due to every single individual with the Miller-Dieker syndrome, also identified as having a mutation within this region.

LIS1‘s role in the brain could be due to the interactions it makes with protein complexes which are likely to play a role in brain development as is the case in many WD repeat containing proteins. LIS1 functions as a dimer in all the organisms studied so far, indicating homo-dimerisation is a significant factor in order for it to function correctly. This dimerisation feature is what deviate the protein from the typical WD repeat containing proteins making its function analysis slower than other proteins, as there is not much to compare to. Initial results were, when a truncated version of the LIS1 protein was produced with directed mutagenesis of the first exon, it was observed that the protein could not associate with the PAFAH protein. Interestingly, what this triggered was an increase in the enzymatic activity of the PAFAH protein. However what is even more interesting is that PAFAH does not have an interaction with microtubules (Hirotsune et al., 1998) indicating that LIS1 independently takes part in
different interactions.

So how does the subunit of the PAFAH enzyme – LIS1, affect the brain?

PAFAH functions as the in-activator of the platelet activating factor (PAF) with removal of the acetyl group. PAF, which is an acetylated derivative of glycerophosphorylcholine, plays a role in neuronal differentiation and can also act as an intracellular messenger. The main effect
of it is to induce platelet aggregation. Patients who lack PAF have severe neuronal migration defects. In mouse models the same defects were also found which connected the PAFAH enzyme with brain development. Since LIS1 was known to be playing a part in brain development, PAF – which is a lipid messenger known to play a role in the inflammatory process; was also suggested to have a function in the process (Howard et al., 2011).  Strangely no evidence of this has been found so far. Since there is not much that can be done on humans about elucidating the functions and interactions of LIS1, research groups – as often is the case, looked for alternatives; and they have identified homologues of LIS1 in many organisms from slime moulds to fruit flies. Also since it is not possible to study neuronal migration embryologically in humans – a process which LIS1 definitely plays a role in, mouse models have been the most attractive alternatives due to their similarities with humans in development.

LIS1 in other Organisms

The same questions – asked in humans; were also investigated in different organisms about the LIS1 gene. In mice and Drosophila, the protein was seen to be expressed as a maternal component of the oocyte. Furthermore, embryos which were homozygous for the mutated allele died early in development, following embryo implantation in mice and as larvae in fruit flies. The mushroom body, which are involved in learning and memory in Drosophila, have high levels of LIS1 [removed]Siller & Doe, 2008). LIS1 was found to be one of the essential genes in neuroblast proliferation, and LIS1 mutants showed abnormalities in the axonal transport and had shorter dendrites.

As the LIS1 gene is mostly associated with the brain in humans, brain development was the key area of research when investigating LIS1's role in other organisms. Brain structure formation is a very complex process which requires the coordination and interaction of many genes; and LIS1 is one them. Mouse models have enlightened this complex process and gave strong indications that LIS1 is involved with the formation of the brain in some way or another. In mice with reduced expression of LIS1 – although the amount of differentiated neurons was at normal levels; they were randomly distributed throughout the cerebrum
rather than being aligned within their layers as in normally developing mice (reviewed by Vallee et al., 2001). This abnormality in neuron alignment resulted in minimal brain function and uncontrollable seizures, even leading to death occasionally.

Actively dividing epithelial layers form the neurons during cerebral development under normal
conditions. During differentiation, the neurons migrate towards their layer of function. This process requires an extraordinary control mechanisms and cell motility; how it happens at a fine tuning is still not fully understood.

LIS1 is shown to be associated with microtubule based motor proteins in many organisms. Homologs of LIS1 have also been found in organisms such as the nudF (formerly known as mNudE) gene in the fungus Aspergillus nidulans. Mutants of the gene were shown to be defective in nuclear migration (Feng et al., 2000). In Drosophila melanogaster, LIS1 (DLIS1) was shown to be essential for nuclear positioning and germ line division (Lei et al., 2000), where as in Saccharomyces cerevisiae, a LIS1 (ScLIS1) homolog was presented as essential in nuclear migration (Fujiwara et al., 1999). Mutations in the nudF gene caused smaller colonies to be formed. This was found to be due to failure of dynein function. In mammalian cells and tissue extracts, LIS1 was co-immunoprecipitated with dynein and dynactin in molecular analyses, consistent with findings in other organisms.

Microtubules are essential components of the cytoskeleton and are involved in many cellular
processes such as mitosis (and meiosis) and transport of many organelles within the cell. But the microtubules cannot carry out their functions without the motor proteins such as dynein and tubulin. These proteins are required for the segregation of the sister chromatids in mitosis and meiosis. Dynein seems to be the trivial one in neurons due to its roles in intracellular transport and its ability to transmit signals through long distances with respect to their sizes.
It also participates in cell division and in all sorts of cellular trafficking and axonal transport.

DLIS1 which is the homolog of LIS1 in Drosophila has also been under investigation for over 10 years. In hypomorphic DLIS1 mutants, nuclear positioning defects were observed within the oocyte and the photoreceptor cells; which is consistent with what was observed in Aspergillus nidulans. DLIS1 is co-localised with the dynein protein in the posterior region of the oocyte cortex. It is thought that it might be interacting with the microtubules springing from the oocyte nucleus. In DLIS1 mutants, dynein was not detected in the cortex of the flies which indicated LIS1 is also involved in dynein targeting.  In 2000, Liu et al showed that DLIS1 is required (Liu et al., 2000) for neuroblast proliferation, dendritic elaboration and axonal  transport. In mouse brain nuclei remained near the centre of the mutant cell aggregate where as in the wild type mice they were dispersed. Loss of LIS1 resulted in abnormal neuronal formation as well as cortical germinal zone exiting failure, resulting in disrupted production and migration of neurons in the developing brain.

Nude1 lacking mice – a gene presented to interact with LIS1; showed severe aberrations in spindle orientations supporting the hypothesis that LIS1 regulates the division of neuronal precursor cells in the brain (Feng and Walsh, 2004). In Caenorhabditis elegans, LIS1 knockout mutants showed defects in centrosome separation and spindle assembly as well as inactivation of dynein. Complete loss of LIS1 caused lethality.

DdLIS1, an orthologue of LIS1 in the slime mould Dictyostelium discoideum, has also gained great interest of late, due to its surprisingly high DNA sequence identity of 53% (reviewed by Meyer et al, 2011) with LIS1. It also has a similar mass of 47kDa, compared to LIS1's 45kDa. DdLIS1 knockouts were lethal in Dictyostelium also, thus it was studied using hypomorphic mutations. As a result dynein associated functions were perturbed in these mutants  compatible with the mammalian findings. DdLIS1 mutants also showed disrupted microtubule cytoskeleton, Golgi apparatus dispersal and centrosome detachment.

This finding in the DdLIS1, led to further study of LIS1 and dynein. It was found that LIS1 binds to dynein and interacts with the microtubule plus ends, to play its role in important processes such as spindle orientation and nuclear migration during neuronal development – which are crucial processes in correct neocortical patterning (reviewed by Morris N, 2000). 
Nde1, Ndel1, CLIP170 and doublecortin are also involved in these processes (Yan et
al., 2003). Nde1 and Ndel1 are known to be necessary for the membrane association of the LIS1-dynein complex.

Periventricular heterotopia which is a milder form of lissencephaly, is caused by mutations in
genes which encode regulators of the actin cytoskeleton and it is easy to comprehend
why, as actin cytoskeleton plays an essential role in cell locomotion. This finding has led to LIS1 studies to concentrate towards the actin cytoskeleton. Recent studies then indicated that LIS1's interactions do not end with the microtubules but carries onto the actin filaments also. This was first shown by Rehberg et al when LIS1 was shown to interact directly with Rac1A in Dictyostelium (Rehberg et al., 2005), a GTPase which regulates the actin cytoskeleton. The Rac1a – through activation of the actin bundling proteins cortexillin 1 and cortexillin 2;  regulates the cortical actin cytoskeleton. There are several suggestions as to how LIS1 acts in this pathway, the most regarded one being that LIS1 is involved in Rac1a activation or in the proteins delivery to its site of action. This is supported by the findings in DdLIS1 overexpressing Dyctyostelium, where DdLIS1 localised indistinguishably with Rac1a.

In 2006, Kholmanskikh and his coworkers published a putative role for LIS1 in ρ (Rho)  GTAse regulation (Kholmanskikh et al., 2006). They presented that in mice cells, LIS1 could be acting via a Ca2+ sensitive GTPase scaffolding protein named IQGAP when interacting with the cortical actin and microtubule ends. They showed cdc42 – a protein which is involved in regulation of the cell cycle; was activated when LIS1 and IQGAP interacted. This has led to several models including the Ca2+ influx model, which states that IQGAP is released from calmodulin when Ca2+ levels increase, and promote its interactions with Rac1a and cdc42 via interaction with LIS1 (Kholmanskikh et al., 2003). However, further study is needed on this role of LIS1.

Doublecortin – an X linked gene in humans; as mentioned above is another protein LIS1
interacts with. However no doublecortin-like kinase is present in the Dictyostelium genome thus it is likely that DdLIS1 compensates for it in the slime mould, since both of them act in the same pathways in mammals (Caspi et al., 2000) and this is likely to be the case in the Dictyostelium. Interestingly, overexpression of doublecortin eliminates cell migration defects in LIS1 heterozygous mutant neurons (Tanaka et al., 2004) in mice, which is the case in Dictyostelium as well. Furthermore doublecortin and DdLIS1 have been shown to work
together in cAMP signalling – important in intracellular signal transduction; suggesting that the two proteins could be playing roles independent of the cytoskeleton. As with most findings about LIS1, further study is needed in this process. Also one of Doublecortin functions is to stabilise the microtubules. Mutations in the doublecortin gene, affects the ventral side of the brain (Gleeson et al., 1999) but why this happens still needs elucidating, although it is thought to be through the actin cytoskeleton pathway.

Different paths are also taken to identify LIS1's roles, such as overexpression.

LIS1 Overexpression

A recent article published by Bi and his coworkers has great relevance for LIS1 research as a new approach was taken while joining the search for more answers about LIS1.  How  overexpressing LIS1 affected mice was the answer they were looking after, which was not tried before. After all, beside new information about previously known roles of LIS1, a potentially new role for the gene was also published; a role for LIS1 in cell polarity. There were many studies about mutations within and complete knockouts of LIS1 gene  (PAFAH1B1) and its effects, but not many about how overexpression affected humans
as well as mice. This study showed that LIS1 duplication (and triplication) caused change in brain size (Bi et al., 2009) in humans and mouse. The brain MRI scan of two individuals showed significant decrease in brain size; more in the individual with triplication of the PAFAH1B1 gene than the individual with a duplication. This led to a hypothesis that the more copies of the LIS1 gene, smaller the size of the brain. It is a very interesting prediction and how things will plan out in the future will be eagerly awaited.

Moving on to mice which had LIS1 was overexpressed 20% more than control mice; they had significantly smaller brain size (Bi et al., 2009). Most reduction occurred at the somatosensory cortex. Analysis of the brain sections also showed disorganisation in the ventricular zone. Progenitors which normally divide at apical positions within the ventricular zone were widely distributed in LIS1 overexpressing mice (Bi et al., 2009). Interestingly higher proportions of phosphorylated histone H3 were observed compared to control mice in LIS1 overexpressing mice. Nestin, which is an intermediate filament protein was also expressed more in LIS1 overexpressing mice compared to control; and finally more apoptotic cells were observed in the former. The latter is a completely new finding and how LIS1 affects the apoptotic pathway is completely unknown at present.

A role in cell polarity was another putative role of LIS1 published by Bi and his co-workers. Polarity is critical for neuro-epithelial cells in the ventricular zone of the brain. To analyse how LIS1 overexpression affected cell polarity, analyses were carried out in the E14.5 section of the brain using immunostaining. Their results have revealed reduction in cell polarity and disruptions in apical junctions.  

Beta-catenin, which is a subunit of the cadherin protein complex, was found to be distributed throughout the cell in LIS1 overexpressing mice whereas it was located in punctate structures near the ventricle in normal mice. Also centrosomes which are tethered to the apical surface in normal mice were scattered in LIS1 overexpressing mice.

Finally, LIS1 was known to play a role in neuronal migration but how overexpression affected this process was not very well known. They illustrated that LIS1 overexpressing mice showed delay in pre-plate splitting thus the cortical plate was not formed at normal time period relative to control mice (Bi et al., 2009).

Conclusion

The LIS1 gene and its product seem to be one of those which have a hand in any processes. This is probably the main reason why it has taken, and still is taking; so much time and effort while trying to find its functions and interactions. Over the last 20 years or so, research  groups have tried all sorts of techniques from mutating the gene to completely deleting it to deduce the pathways it affects and/or regulates. While some of them have been in vain, others have been very useful, particularly the mice models. In mice, LIS1 was identified as having a role in a wide range of pathways, most striking ones being the microtubule organisation and neuronal migration; as these interactions gave better ideas as to why mutations in LIS1 were affecting the brain – as this phenomenon in humans had puzzled scientists for years.

LIS1 shows deviations from typical WD repeat containing proteins adding to the difficulties already being faced as there is not much to compare LIS1 to. Every single one of alleged
roles of LIS1 at present – whether a lot or less; still needs further analysis. No paper is present at the moment, which talks conclusive about even a single role of LIS1.

All studies done until now show that either one of: (i) mutations in LIS1 (ii) reduced expression of LIS1 or (iii) overexpression of LIS1 can produce a range of disorders in humans and other organisms. Findings have suggested that LIS1 could be affecting neuronal migration by interfering with mitosis and reducing the number of migratory cells or by directly interfering with the cytoskeletal dynamics, which requires deeper analysis.

LIS1 homologs have been shown to play critical roles in regulating nuclear movement
in organisms ranging from fungi to mammals. Mitotic spindle orientation and chromosome segregation are also a couple of the many processes LIS1 delves into. However the most
intriguing ones are the potential role of LIS1 in apoptosis, histone phosphorylation and cell polarity, which all require further analysis to say anything definitive. Hypotheses on these processes are being changed and adjusted frequently, as knowledge accumulates about the LIS1 gene and its functions.

Elucidation of the neuronal migration process and LIS1 role in these finely tuned processes can lead to better understanding of the repopulation process of the brain from stem-cell
precursors and even the invasiveness of tumour cells in the brain, which would be a massive finding for mankind. Also some parts of the body seem to be more sensitive – such as the brain; to LIS1 dosage than others and why this is happening adds to the tens of questions
still waiting to be answered about LIS1.

References

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