Functional Analysis of Schizophrenia Risk Genes: Complements on an Elegant Piece of Work

Schizophrenia (see here) is a debilitating disorder comprising of three symptom domains: 1. The so-called positive (psychotic) symptoms of auditory hallucinations (hearing voices), paranoia and disorganised behaviour; 2. Negative symptoms, such as a lack of motivation and the loss of pleasure in activities that were formerly pleasurable; and 3. Cognitive deficits, reflected by a lack of mental agility and a general “brain fog”. Although the positive symptoms are relatively well-controlled by first- and second-generation antipsychotic drugs, such as haloperidol, risperdol, olanzapine, paliperidone, and aripiprazole, all of which antagonise dopaminergic D2 receptors, the negative symptoms and cognitive deficits are poorly treated. Unfortunately, the search for new treatments for schizophrenia is hampered by the lack of an understanding of the underlying pathological mechanisms. However, recent genetic data has begun to shine light onto some of the mysteries of the disorder and have implicated a role for complement component 4 (C4) genes (see here).

Prior to the advent of genome-wide association (GWA) studies, candidate gene studies implicated a number of different genes in the pathogenesis of schizophrenia (such as COMT, DISC1, DTNBP1 and NRG1) yet due to a lack of statistical power, these have failed to provide meaningful insights into the pathology of the disease (see here). More recently, the use of genome-wide association (GWA) studies, which links disease risk to specific regions of the genome, has begun to identify genetic variants associated with schizophrenia. Hence, the Schizophrenia Working Group of the Psychiatric Genomics Consortium reported at least 108 different regions of the genome associated with a risk of schizophrenia (see here). These data highlighted associations between the dopamine D2 receptor and genes involved in glutamate neurotransmission, consistent with the well-described dopamine and glutamate dysfunction hypotheses of schizophrenia. Intriguingly, a number of other genes were associated with the immune system, of which the strongest were those associated with the major histocompatibility complex (MHC), a region containing 18 polymorphic human leukocyte antigen (HLA) genes. The genetic links between the immune system and schizophrenia is consistent with epidemiological data suggesting, for example, that over a third of cases could be prevented if infection in pregnant women was prevented (see here).

Fig 1Complex association

The MHC is a complicated region of Chromosome 6 and is divided into three classes (MHC I, II and III; see figure 1, taken from here). Recent work from Sekar and colleagues (see here) has focussed upon the most strongly associated markers in the Class III region and more specifically the C4 gene. The C4 gene has the added complexity of existing as C4A and a C4B isotypes, both of which vary in structure and copy number and both have a long and short form, differentiated by the present or absence, respectively, of a human endogenous retroviral (HERV) insertion, which lengthens the gene but not the protein sequence. To cut a long and complicated story short, the different C4 alleles resulted in widely varying levels of C4A and C4B expression in the brain (Figure 2 – see Sekar et al) with greater levels of C4A expression being related to a greater risk of schizophrenia.


Figure 2. Expression levels of C4A RNA


In the immune system, C4 activates C3, which in turn covalently attaches to its targets and triggers engulfment by phagocytic cells and in the developing mouse brain, C3 is important for synaptic pruning, a crucial process in neurodevelopment. Accordingly, Sekar and colleagues showed that mice deficient in the C4 gene had deficits in synaptic remodelling (reduced synaptic pruning) similar to those observed in C3-deficint mice. Hence, the increased expression of C4 protein in schizophrenia may well result in increased synaptic pruning that might in turn be related to the cortical thinning and reduction in synaptic organisation that have been reported in schizophrenia. Although such studies will not directly lead to novel therapeutics, they nevertheless demonstrate an elegant translation of genetic information into functional studies and provide the basis for hypotheses that might possibly transform the treatment of this disorder.


Blog writted by John Atack


DNA damage and repair: Let’s use our brains!

The integrity of our DNA is under constant attack from numerous endogenous and exogenous agents. The consequences of defective DNA and DNA damage responses (DDRs) have been extensively studied in fast proliferating cells, especially in connection to cancer, yet their precise roles in the nervous system are relatively poorly understood.

Two fundamental questions are still open:

What is the integrity of the genome in the adult and aging brain?

 What is the role of DNA damage in aging and neurodegenerative disorders such as Alzheimer’s disease (AD) or Parkinson’s disease (PD)?

How damaged is the genome in the adult brain?

The neurons of our nervous system are post-mitotic, meaning that once matured, they cannot rely on cell division to replace a lost or disabled neighbour. This fact has two important consequences:

  • In a long-lived species such as homo sapiens, a ‘lucky’ CNS neuron may survive for 80 years or more, potentially accumulating a lot of DNA damage.
  • Neurons are devoid of homologous recombination – the most effective way to repair DNA double stranded breaks -which takes place mainly during cell division.

The genome integrity in the adult brain is still the object of intense scrutiny but what is generally accepted is that the adult brain tolerates an unexpected degree of DNA damage and that the DDR mechanisms might be significantly different from other somatic cells.

What is the role of DNA damage in aging and neurodegenerative disorders?

As we have already observed, neurons are particularly prone to accumulating DNA defects with age, the key question here is whether these defects contribute to developing and/or sustaining neurodegeneration.

Several pieces of evidence seem to point in this direction. For example, age is the most common risk factor for most adult-onset neurodegenerative diseases with even the most aggres­sive familial forms of dementia rarely striking before the age of 40 years.

What is still unclear is how DNA damage contributes to the development of pathologies such as AD and PD that are regional by nature, e.g. involve only specific areas of the brain.

On this regard, several models and theories have been suggested but none of them has been fully validated yet with sufficient data (Fig.1).

Fig.1 Models to explain the relationship between age, DNA damage and neurodegeneration. (from: Chow, H-m; Herrup, K. Genomic integrity and the ageing brain’, NATURE REVIEWS NEUROSCIENCE, 2015; 16, p 672)

Alessandro 8-12-2015 Figure 1

DNA damage and the onset of specific neurodegenerative diseases. a | As we age, all of our neurons experience increasing amounts of irreparable DNA damage. The accumulating damage is induced by products of cell metabolism and other destructive activities (black arrows) coupled with a reduced capacity for DNA repair (grey arrows). Disease initiation then arises as a result of an additional insult, specific to the particular degenerative condition, which, coupled with the damage already present, precipitates the emergence of disease. Without that insult, a slow but benign descent into ageing would continue without serious clinical consequences (as indicated by the dashed line). Once the activity of DNA repair can no longer keep pace with the rate at which DNA damage is generated, damage accumulates at an increased pace and a point of no return is reached, eventually leading to neuronal death. b | An alternative, but not mutually, exclusive conceptualization involves a network-based model of DNA damage. If the relative activity levels of different circuits of neurons leads to the accumulation of specific unrepaired DNA lesions in the participating cells 42 , the predicted consequence would be regional variability in the rates of DNA damage, leading to different rates of neuronal ageing and hence to specific selections of neurodegenerative events. For instance, during the development of Alzheimer disease (AD), aberrant activities of neurons in the hippocampal network might result in the lethal accumulation of DNA damage in certain cells. Within the same brain, Purkinje cells in the cerebellum, engaged in a different pattern of physiological activity, would show minimal accumulation of such damage and be spared. After many years, the loss of genomic integrity in the most affected hippocampal neurons would lead to a pattern of cell dysfunction and death that would be more pronounced than that in the cerebellum. A similar branching network model with different initiation points could be envisioned for other diseases, including Parkinson disease (PD), Lewy body disease (LBD) and epilepsy.


Clearly, answering to some of these questions could open new exciting avenues in the field of neurodegeneration, an area that unfortunately is increasingly neglected by big pharma after the clinical failures of the last decade.

It is definitely time for DDR research to focus on the brain!

Blog written by Alessandro Mazzacani

Further reading:

‘Genomic integrity and the ageing brain’, Hei-man Chow and Karl Herrup, NATURE REVIEWS NEUROSCIENCE, 16, NOVEMBER 2015, p 672

 DNA Damage and Its Links to Neurodegeneration’ Ram Madabhushi, Ling Pan,and Li-Huei Tsai, Neuron, 83, July 2014, p 266

Gli1 – a brand new drug target for demyelinating disorders?

The elucidation of a pathway involved in remyelination has yielded a novel potential target in the treatment of neurodegenerative diseases such as multiple sclerosis (MS).

MS is an autoimmune neurological condition affecting around 100,000 people in the UK alone and is characterized by demyelination – damage to the insulating myelin sheath of neurons. MS causes a wide range of symptoms depending on the location of the damage in the CNS and can include fatigue, blurred vision, mobility problems and muscle weakness, often drastically decreasing quality of life. Many current treatments target the immune system in an attempt to slow the disease, but drugs that have the ability to combat demyelination itself have so far remained elusive. Now, a series of elegant experiments by researchers at NYU Neuroscience Institute ( appears to have shed some light on the process of endogenous remyelination, which has important implications for the treatment of demyelinating disorders.

In adult human and mouse brains, remyelination is performed by two cell types: oligodendrocyte progenitor cells (OPCs) and neural stem cells (NSCs). Little is known about how NSCs in particular are recruited to lesion sites for their ultimate differentiation into oligodendrocytes, but a likely candidate involved in their regulation is sonic hedgehog (Shh), a morphogen with important roles in CNS development and NSC maintenance. Specifically it is Gli1, a transcription factor downstream in the Shh signaling pathway that has been the focus of this research.

Studying mice that express green fluorescent protein (GFP) in all Gli1-expressing cells, the researchers were first able to identify that Gli1+ NSCs have a prominent role in remyelination, and continue to generate glial cells for a prolonged period of time after demyelination. Using cuprizone to stimulate selective demyelination in the corpus callosum (CC), it was observed that GFP-expressing cells were recruited to damaged areas after six weeks, which then differentiated exclusively into glial cells – primarily oligodendroglia – two weeks after cuprizone was removed. Further, the numbers of GFP+ cells – comprising of OPCs, oligodendrocytes and astrocytes – continued to increase ten weeks after cuprizone removal.

Gli1-expressing cells are recruited to sites of demyelination. Markers of oligodendrocytes (CC1), oligodendrocyte progenitors (PDGFR-alpha) and astrocytes (GFAP) are observed in increasing amounts after cessation of cuprizone.

The relationship between Shh and Gli1 was then probed by further fate-mapping experiments of Gli1+ cells in mice, and it was found that Shh-responsive NSCs are recruited to demyelinated lesions in the CC where they downregulate Gli1 and differentiate into mature oligodendrocytes. Based on this finding, the question was raised as to whether inhibition of Shh signaling might enhance remyelination. To this end, the researchers fate-mapped NSCs in Gli1-null mice and indeed found an increase in GFP+ cells in the CC and an overall increase in myelin, but interestingly targeted ablation of Shh signaling did not increase GFP+ cells in the CC, indicating Gli1 has a specialized role in Shh signaling and myelination.

The next logical step, then, was to attempt targeted inhibition of Gli1 as a novel means to promote remyelination. This was done using the experimental drug GANT61, a small molecule inhibitor of Gli1, originally identified from a National Cancer Institute of chemical compounds as a potential therapy for brain and basal cell cancers.

Excitingly, it was found that GANT61 strongly promoted remyelination by enhancing the recruitment and differentiation of Shh-responsive NSCs into oligodendrocytes at demyelinated lesions. Specifically, mice receiving GANT61 during and after cuprizone treatment had significantly greater GFP+ cells, a significant amount of which were oligodendrocytes, resulting in more myelin present in the CC of treated mice compared to control-treated mice. In addition, when GANT61 was tested in a relapsing-remitting model of experimental autoimmune encephalitis (RR-EAE), a model of inflammatory demyelination and remyelination, they observed enhanced levels of remyelination and neuroprotection.

Mice given GANT61 had an approx. sevenfold increase in GFP-labeled cells in the CC (a) and a significant increase in the number of mature oligodendrocytes (b). Mice fed cuprizone then treated with either vehicle or GANT61 showed significantly higher fluorescence levels in GANT61-treated mice (c).

The implications of these findings are twofold: we now have an improved understanding of the mechanisms underlying myelination, and a starting point for the next generation of demyelination-targeting MS drugs. With an increasingly unmet need for well-tolerated MS therapies that do not target the immune system, it would not be surprising if this paves the way for numerous novel drug discovery avenues.
Blog written by Chloe Koulouris

Novel Rat model for Alzheimer’s disease

It is stating the obvious that having good animal models is critical to the success of any drug discovery program. In many more complex diseases however, good animal models are not available. The ‘gold standard’ animal models for Alzheimer’s disease, Aβ-overproducing transgenic AD mice; do not demonstrate robust tauopathy and subsequent neuronal loss without the addition of genes not linked to familial AD.

In a recent paper Cohen et al., (1) have generated transgenic rats bearing human mutant APP (amyloid precursor protein) and PS1 (presenilin 1). These animals appear to manifest the full spectrum of age-dependent Alzheimer’s disease pathologies alongside cognitive disturbances. They have age-dependent β-amyloid deposition as well as intraneuronal Aβ1-42 and soluble Aβ oligomers. Many mouse models do present with some tauopathy, however, they do not present with neurofibrillary tangles (NFT) as observed in human AD. In this rat model however, they identified striking tauopathy. As well as hyperphosphorylated Tau, structures reminiscent to NFTs were identified close to β-amyloid plaques in aged rats. In addition immunostaining revealed structures consistent with NFTs in 16 month old rats. These NFT-like structures were also frequently observed in areas without plaques, as is found in human AD.

In concert with the molecular pathology, these transgenic rats exhibited neuronal loss and neuronal degeneration that was progressive and age-dependent. There was also an inverse correlation between the neuronal numbers and Aβ1-42 abundance. TUNEL staining indicated the presence of nicked DNA and measurements of active caspase-3 suggested the neurons were apoptosing.  This neuronal loss paralleled changes in behavioural characteristics such as novel object recognition (which is a hippocampal-dependent measure of working memory) that was significantly impaired in older transgenic animals. This was repeated in the Barnes maze, where there were no difference between wild-type and transgenic animals at 6 months, but after 15 months the transgenic animals made significantly more errors than wild-type.

With recent late-stage failures of treatments for Alzheimers this new animal model opens up the possibility to test novel therapeutics in a more human disease-like model.

Eph in ALS

A recent publication from Van Hoecke and colleagues (Van Hoecke et al., 2012) suggests a novel therapeutic approach to the treatment of amyotrophic lateral sclerosis (ALS; also called Lou Gehrig’s disease). ALS is a progressive degenerative disorder that affects 1-2/100,000 people per year and results in death, normally by respiratory failure, 3-5 years after onset. It is caused by a loss of the motor neurons that control muscle movement. There is a hereditary component in about 10% of all ALS cases and in these familial ALS subjects, a variety of genes have been implicated (Al Chalabi et al., 2012), including SOD1, TARDBP and FUS and which encode superoxide dismutase 1 (SOD-1; which was the initial gene reported to be associated with familial ALS in 1993) TAR DNA binding protein (TDP-43) and the Fused in Sarcoma protein, respectively. Recently, a hexanucleotide repeat expansions that occurs in the chromosome 9 open-reading frame 72 gene (C9ORF72) has been described to be associated with ALS with frontotemporal dementia (DeJesus-Hernandez et al, 2011; Renton et al, 2011).

The huge advances in our understanding of the genetics underlying the familial form of ALS have yet to result in breakthrough therapies for this disorder and Riluzole remains the only FDA-approved treatment for ALS. It was approved in 1995 on the basis of clinical studies that demonstrated that it increased survival times in patients, yet the effects are relatively modest and there is a clear need for new and improved treatments for ALS. Since Riluzole was approved, there have been over 30 clinical trials of new treatments but for a variety of reasons (including poor clinical trial design and drug delivery or dose selection issues) none have reached the market, although dexpramipexole, which enhances mitochondrial function, is currently undergoing Phase III trials sponsored by Knapp (Cudcowicz et al, 2011).

A key challenge to the development of new drugs based upon the genetic information derived from familial ALS, as well as genes associated with sporadic ALS, is to understand how mutations in the various genes produce a similar clinical and pathological phenotype. In other words, what is the final common pathway by which these genetic mutations produce ALS? Generic explanations such as mitochondrial dysfunction or alterations in protein degradation pathways have been suggested but how these processes are affected by genetic influences remain vague. However, it is not necessary to understand the mechanism if one can develop a screen that rescues the phenotype produced by different mutations, and this is what Van Hoecke and colleagues did. Hence, they screened for different morpholinos (antisense oligos in which ribose or deoxyribose is replaced by a morpholine ring) that rescued a SOD-1 induced axonopathy in zebra fish. The most protective morpolino targeted the zebra fish Rtk2 gene, which has 67% identity to the human EPHA4 gene that encodes for the Epha4 receptor tyrosine kinase that can bind both type A and type B ephrins. Knock down of the Rtk2 gene rescued the phenotype in zebra fish with various SOD1 mutants (A4V, G37R and G93A) and SOD-1-induced axonopathy could also be rescued pharmacologically by inhibition of Epha4 using 2,5-dimethylpyrrolyl benzoic acid.  Importantly, knockdown of Rtk1, which is a paralog with 83% identity to human Epha4, was able to rescue the axonopathy induced by either mutant SOD-1, TDP-43 or knockdown of Smn1 in zebra fish, indicating that inhibition of EphA4 is protective against motor neuron degeneration irrespective of the genetic determinant of vulnerability. Having identified Epha4 as a potential modifier of SOD1-mediated pathology, the authors also studied the effects of a deletion of the Epha4 gene in mice overexpressing the G93A mutant SOD1 and were able to show that in heterozygotes, a 50% reduction in Epha4 was able to prolong survival.

As regards ALS itself, EphA4 mRNA expression in total blood was inversely collected to the age of onset such that patients with lower levels of EphA4 expression had an age of onset older than those with higher levels of expression. Suggesting that reduced EphA4 expression is associated with a reduced disease severity. Collectively, these data shed light onto an intriguing pathway in which rescue of the axonopathy is achieved irrespective of the genetic cause. A further understanding of the mechanism by which Epha4 exerts these effects could provide the basis for novel therapeutic approaches to treating ALS.


Al-Chalabi, A., Jones, A., Troakes, C., King, A., Al-Sarraj, S. and van den Berg, L.H. (2012) The genetics and neuropathology of amyotrophic lateral sclerosis. Acta Neuropathol., 124:339-352.

Cudkowicz, M., Bozik, M.E., Ingersoll, E.W., Miller, R., Mitsumoto, H., Shefner, J., Moore, D.H., Schoenfeld, D., Mather, J.L., Archibald, D., Sullivan, M., Amburgey, C., Moritz, J. and Gribkoff, V.K. (2011) The effects of dexpramipexole (KNS-760704) in individuals with amyotrophic lateral sclerosis. Nat. Med., 17:1652-1656.

DeJesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., et al. (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron, 72:245-256.

Renton, A.E., Majounie, E., Waite, A., et al., A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron, 72:257-268.

Van Hoecke, A., Schoonaert, L., Lemmens, R., Timmers, M., Staats, K.A., Laird, A.S., Peeters, E., Philips, T., Goris, A., Dubois, B., Andersen, P.M., Al-Chalabi, A., Thijs, V., Turnley, A.M., van Vught, P.W., Veldink, J.H., Hardiman, O., Van Den Bosch, L., Gonzalez-Perez, P., Van Damme, P., Brown, R.H. Jr., van den Berg, L.H. and Robberecht, W. (2012) EPHA4 is a disease modifier of amyotrophic lateral sclerosis in animal models and in humans. Nat. Med., Aug 26. doi: 10.1038/nm.2901. [Epub ahead of print]