The Role of CNTNAP2 in the Development & Evolution of the Human Cerebral Cortex

The differing cognitive abilities of humans and other primates are accompanied by changes to forebrain neuronal circuit composition and function. This is underpinned by species-specific features of their development. Excitatory neurons in the human cerebral cortex have longer neurites and more elaborate neurite branching than chimpanzees or macaques, which is thought to contribute to differences in neural network performance. To study how differences in expression of single genes contributes to inter-species differences in neuronal form and function, my research focused on Contactin-Associated Protein-like 2 (CNTNAP2), a gene important for neuronal differentiation and synapse formation, for which there is accumulating evidence for differential use in the human cerebral cortex compared with other primates. Previous work in mice has found that loss of Cntnap2 function reduces neurite branching and dendritic spine density in vitro and in vivo. The potential evolutionary significance of this gene is highlighted by the presence of eight human accelerated regions (HARs), suggesting that there are human-specific aspects to its temporal and spatial expression during cortical development. The research presented in this thesis used human and non-human primate stem cell- derived forebrain neurons to study several aspects of CNTNAP2’s function in human cerebral cortex development. We present the first study of neurite outgrowth and neuronal activity in forebrain neurons generated from a human CNTNAP2 knockout (KO) pluripotent stem cell line. Differentiated human CNTNAP2 KO neurons have reduced neurite branching relative to wild type cells. Strikingly, the KO neurons were significantly more active - bursting more strongly and more frequently. We also applied a combination of bioinformatic and experimental approaches to show one or more of the CNTNAP2 HARs may be a gene enhancer. Our results indicate that loss of function mutations in CNTNAP2 contribute to human neurodevelopmental diseases through altering neuronal activity. This may be due to changes in neurite branching, and ultimately, to neuronal connectivity. These discoveries suggest a mechanism by which CNTNAP2 mutations causes disease in affected children, and may have contributed to the evolution of human-specialized brain function. This model will now be invaluable in deciphering the downstream molecular events caused by CNTNAP2 mutations, and may provide molecular targets for novel treatments in CNTNAP2 patients

The role of contactin-associated protein-like 2 in neurodevelopmental disease and human cerebral cortex evolution

The contactin-associated protein-like 2 (CNTNAP2) gene is associated with multiple neurodevelopmental disorders, including autism spectrum disorder (ASD), intellectual disability (ID), and specific language impairment (SLI). Experimental work has shown that CNTNAP2 is important for neuronal development and synapse formation. There is also accumulating evidence for the differential use of CNTNAP2 in the human cerebral cortex compared with other primates. Here, we review the current literature on CNTNAP2, including what is known about its expression, disease associations, and molecular/cellular functions. We also review the evidence for its role in human brain evolution, such as the presence of eight human accelerated regions (HARs) within the introns of the gene. While progress has been made in understanding the function(s) of CNTNAP2, more work is needed to clarify the precise mechanisms through which CNTNAP2 acts. Such information will be crucial for developing effective treatments for CNTNAP2 patients. It may also shed light on the longstanding question of what makes us human

Loss of CNTNAP2 Alters Human Cortical Excitatory Neuron Differentiation and Neural Network Development

BACKGROUND: Loss-of-function mutations in the contactin-associated protein-like 2 (CNTNAP2) gene are causal for neurodevelopmental disorders, including autism, schizophrenia, epilepsy and intellectual disability. CNTNAP2 encodes CASPR2, a single-pass transmembrane protein that belongs to the neurexin family of cell adhesion molecules. These proteins have a variety of functions in developing neurons, including connecting presynaptic and postsynaptic neurons, and mediating signalling across the synapse. METHODS: To study the effect of loss of CNTNAP2 function on human cerebral cortex development, and how this contributes to the pathogenesis of neurodevelopmental disorders, we generated human iPSCs from one neurotypical control donor null for full-length CNTNAP2, modelling cortical development from neurogenesis through to neural network formation in vitro. RESULTS: CNTNAP2 is particularly highly expressed in the first two populations of early-born excitatory cortical neurons, and loss of CNTNAP2 shifted the relative proportions of these two neuronal types. Live imaging of excitatory neuronal growth showed that loss of CNTNAP2 reduced neurite branching and overall neuronal complexity. At the network level, developing cortical excitatory networks null for CNTNAP2 had complex changes in activity compared to isogenic controls: an initial period of relatively reduced activity compared with isogenic controls, followed by a lengthy period of hyperexcitability, and then a further switch to reduced activity. CONCLUSIONS: Complete loss of CNTNAP2 contributes to the pathogenesis of neurodevelopmental disorders through complex changes in several aspects of human cerebral cortex excitatory neuron development that culminate in aberrant neural network formation and function

Does Soluble TREM2 Protect Against Alzheimer's Disease?

Triggering Receptor Expressed in Myeloid Cells 2 (TREM2) is a pattern recognition receptor on myeloid cells, and is upregulated on microglia surrounding amyloid plaques in Alzheimer's disease (AD). Rare, heterozygous mutations in TREM2 (e.g., R47H) increase AD risk several fold. TREM2 can be cleaved at the plasma membrane by metalloproteases to release the ectodomain as soluble TREM2 (sTREM2). Wild-type sTREM2 binds oligomeric amyloid beta (Aβ) and acts as an extracellular chaperone, blocking and reversing Aβ oligomerization and fibrillization, and preventing Aβ-induced neuronal loss in vitro. Whereas, R47H sTREM2 increases Aβ fibrillization and neurotoxicity. AD brains expressing R47H TREM2 have more fibrous plaques with more neuritic pathology around these plaques, consistent with R47H sTREM2 promoting Aβ fibrillization relative to WT sTREM2. Brain expression or injection of wild-type sTREM2 reduces pathology in amyloid models of AD in mice, indicating that wild-type sTREM2 is protective against amyloid pathology. Levels of sTREM2 in cerebrospinal fluid (CSF) fall prior to AD, rise in early AD, and fall again in late AD. People with higher sTREM2 levels in CSF progress more slowly into and through AD than do people with lower sTREM2 levels, suggesting that sTREM2 protects against AD. However, some of these experiments can be interpreted as full-length TREM2 protecting rather than sTREM2, and to distinguish between these two possibilities, we need more experiments testing whether sTREM2 itself protects in AD and AD models, and at what stage of disease. If sTREM2 is protective, then treatments could be designed to elevate sTREM2 in AD.</jats:p

Inherited and Sporadic Amyotrophic Lateral Sclerosis and Fronto-Temporal Lobar Degenerations arising from Pathological Condensates of Phase Separating Proteins.

Recent work on the biophysics of proteins with low complexity, intrinsically disordered domains that have the capacity to form biological condensates has profoundly altered the concepts about the pathogenesis of inherited and sporadic neurodegenerative disorders associated with pathological accumulation of these proteins. In the present review, we use the FUS, TDP-43 and A11 proteins as examples to illustrate how missense mutations and aberrant post-translational modifications of these proteins cause amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD).Supported by Canadian Institutes of Health Research, Wellcome Trust, Zenith Award from the US Alzheimer Society, ALS Society of Canada/Brain Canada

Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition.

Membrane-less organelles resulting from liquid-liquid phase separation of biopolymers into intracellular condensates control essential biological functions, including messenger RNA processing, cell signalling and embryogenesis1-4. It has recently been discovered that several such protein condensates can undergo a further irreversible phase transition, forming solid nanoscale aggregates associated with neurodegenerative disease5-7. While the irreversible gelation of protein condensates is generally related to malfunction and disease, one case where the liquid-to-solid transition of protein condensates is functional, however, is that of silk spinning8,9. The formation of silk fibrils is largely driven by shear, yet it is not known what factors control the pathological gelation of functional condensates. Here we demonstrate that four proteins and one peptide system, with no function associated with fibre formation, have a strong propensity to undergo a liquid-to-solid transition when exposed to even low levels of mechanical shear once present in their liquid-liquid phase separated form. Using microfluidics to control the application of shear, we generated fibres from single-protein condensates and characterized their structural and material properties as a function of shear stress. Our results reveal generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. These observations suggest that shear can play an important role in the irreversible liquid-to-solid transition of protein condensates, shed light on the role of physical factors in driving this transition in protein aggregation-related diseases and open a new route towards artificial shear responsive biomaterials

Prevention of Alzheimer's disease in high risk groups: statin therapy in subjects with PSEN1 mutations or heterozygosity for apolipoprotein E epsilon 4

Because cerebrospinal fluid (CSF) abnormalities in presymptomatic subjects with PSEN1 (presenilin 1) mutations may be observed 4 to 12 years prior to the estimated age at onset, it is possible to test putative therapies on the CSF analytes that correlate with neurodegeneration during this presymptomatic window of clinical opportunity. It is also possible to test the same therapy on a comparison group with increased risk status conferred by both hyperlipidemia and heterozygosity for apolipoprotein Eε4. To our knowledge, the only putative therapy thus far tested in such a common design has been statin therapy. The results of these tests show increases in soluble amyloid precursor protein (sAPP)α correlating with statin-induced decreases in serum cholesterol levels in the non-PSEN1 subjects. This result could be one functional correlate for part of the substantial risk reduction for late onset Alzheimer\u27s disease recently reported in the Rotterdam study, a large, long-term prospective statin trial. Statin therapy significantly decreased both sAPPα and sAPPβ in presymptomatic PSEN1 subjects. Initially, elevated phospho-tau levels in PSEN1 subjects did not further increase during the 2 to 3 years of statin therapy, possibly indicative of a prophylactic effect. These results suggest that large and longer term trials of statin therapy correlating changes in CSF biomarker levels with clinical course may be warranted in both presymptomatic PSEN1 and non-PSEN1 subjects

Identification of Small Molecule Inhibitors of Tau Aggregation by Targeting Monomeric Tau As a Potential Therapeutic Approach for Tauopathies.

A potential strategy to alleviate the aggregation of intrinsically disordered proteins (IDPs) is to maintain the native functional state of the protein by small molecule binding. However, the targeting of the native state of IDPs by small molecules has been challenging due to their heterogeneous conformational ensembles. To tackle this challenge, we applied a high-throughput chemical microarray surface plasmon resonance imaging screen to detect the binding between small molecules and monomeric full-length Tau, a protein linked with the onset of a range of Tauopathies. The screen identified a novel set of drug-like fragment and lead-like compounds that bound to Tau. We verified that the majority of these hit compounds reduced the aggregation of different Tau constructs in vitro and in N2a cells. These results demonstrate that Tau is a viable receptor of drug-like small molecules. The drug discovery approach that we present can be applied to other IDPs linked to other misfolding diseases such as Alzheimer's and Parkinson's diseases.We thank the Wellcome Trust (UK), Medical Research Council (UK), Elan Pharmaceuticals (USA), the Canadian Institutes of Health Research (Canada) and the Alzheimer Society of Ontario (Canada), and Hungarian Brain Research Program (KTIA_NAP_13-2014-0009) for funding.This is the author accepted manuscript. The final version is available from Bentham Science via http://dx.doi.org/10.2174/15672050120915101910495