Md. Mahiuddin Ahmed

Pathways to cognitive deficits in Down syndrome.

Major efforts in Down syndrome (DS) research have been directed at the identification and functional characterization of genes encoded by human chromosome 21 (HSA21). In parallel with this, tissue samples and cell lines derived from individuals with DS have been examined for abnormalities in gene expression and cellular morphology, and mouse models of DS have been characterized for abnormalities at the molecular, cellular, electrophysiological, and behavioral level. One goal of such investigations has been the identification of effective targets for pharmacotherapies that can prevent or correct the abnormalities and, by extension to human clinical trials, prevent or lessen aspects of the cognitive deficits seen in people with DS. Because it is caused by an extra copy of an entire chromosome, DS has been considered by some as too complicated a genetic perturbation to be amenable to postnatal pharmacological interventions. However, recent data from experiments with one mouse model, the Ts65Dn, have clearly demonstrated that several pharmacological interventions can indeed rescue DS-relevant learning and memory deficits. Extension of mouse data to successful human clinical trials will be aided by understanding the molecular basis of successful drug treatments, that is, how increased expression of HSA21 genes perturbs molecular mechanisms that are targeted and rescued by specific drugs. Here, we review information on HSA21 genes, their expression and their likely contributions to the DS phenotype. We then describe results of a bioinformatics effort that integrates information on genes known to cause intellectual disability when mutated, the pathways in which these genes function, and how these pathways are impacted by HSA21 encoded proteins. This pathway approach to the molecular basis of ID in DS aids in understanding why some drug therapies have been successful in the Ts65Dn and in predicting whether these same drugs are likely to be successful in treating ID in DS. These data can be used to design new experiments and interpret information for prediction of additional targets for effective drug treatments.

Preserving protein profiles in tissue samples: differing outcomes with and without heat stabilization

Post translational modification (PTM) and proteolytic processing of proteins contributes to regulation of their stability, intracellular localization and interactions with other proteins, and to direct enhancement or repression of their activity. Both PTM and proteolysis are dynamic; levels and rates change in response to changes in the cellular environment. Tissue excision, post mortem interval and subsequent methods of tissue processing for protein analysis unavoidably alter the cellular environment and therefore features of protein profiles. To aid in understanding the time frame and protein specificity of these changes and the biological and technical contributions to them, we have compared features of protein profiles in mouse hippocampus and cortex following three methods of tissue handling: immediate lysate preparation, and rapid heating to 95°C and standard snap freezing in liquid nitrogen, prior to lysate preparation. In spite of the very short time frames involved, we observe protein-specific differences in levels of phosphorylation, general differences in patterns of sumoylation, and specific differences in levels of proteolytic cleavage of calcineurin and the neurotrophin receptor, TRKA. These differences vary with brain region and with post excision time to processing, and highlight the challenges inherent in accurately profiling the in vivo proteome.

Loss of Correlations among Proteins in Brains of the Ts65Dn Mouse Model of Down Syndrome

The Ts65Dn mouse model of Down syndrome (DS) is trisomic for orthologs of 88 of 161 classical protein coding genes present on human chromosome 21 (HSA21). Ts65Dn mice display learning and memory impairments and neuroanatomical, electrophysiological, and cellular abnormalities that are relevant to phenotypic features seen in DS; however, little is known about the molecular perturbations underlying the abnormalities. Here we have used reverse phase protein arrays to profile 64 proteins in the cortex, hippocampus, and cerebellum of Ts65Dn mice and littermate controls. Proteins were chosen to sample a variety of pathways and processes and include orthologs of HSA21 proteins and phosphorylation-dependent and -independent forms of non-HSA21 proteins. Protein profiles overall show remarkable stability to the effects of trisomy, with fewer than 30% of proteins altered in any brain region. However, phospho-proteins are less resistant to trisomy than their phospho-independent forms, and Ts65Dn display abnormalities in some key proteins. Importantly, we demonstrate that Ts65Dn mice have lost correlations seen in control mice among levels of functionally related proteins, including components of the MAP kinase pathway and subunits of the NMDA receptor. Loss of normal patterns of correlations may compromise molecular responses to stimulation and underlie deficits in learning and memory.

Protein dynamics associated with failed and rescued learning in the Ts65Dn mouse model of Down syndrome.

Down syndrome (DS) is caused by an extra copy of human chromosome 21 (Hsa21). Although it is the most common genetic cause of intellectual disability (ID), there are, as yet, no effective pharmacotherapies. The Ts65Dn mouse model of DS is trisomic for orthologs of ∼55% of Hsa21 classical protein coding genes. These mice display many features relevant to those seen in DS, including deficits in learning and memory (L/M) tasks requiring a functional hippocampus. Recently, the N-methyl-D-aspartate (NMDA) receptor antagonist, memantine, was shown to rescue performance of the Ts65Dn in several L/M tasks. These studies, however, have not been accompanied by molecular analyses. In previous work, we described changes in protein expression induced in hippocampus and cortex in control mice after exposure to context fear conditioning (CFC), with and without memantine treatment. Here, we extend this analysis to Ts65Dn mice, measuring levels of 85 proteins/protein modifications, including components of MAP kinase and MTOR pathways, and subunits of NMDA receptors, in cortex and hippocampus of Ts65Dn mice after failed learning in CFC and after learning was rescued by memantine. We show that, compared with wild type littermate controls, (i) of the dynamic responses seen in control mice in normal learning, >40% also occur in Ts65Dn in failed learning or are compensated by baseline abnormalities, and thus are considered necessary but not sufficient for successful learning, and (ii) treatment with memantine does not in general normalize the initial protein levels but instead induces direct and indirect responses in approximately half the proteins measured and results in normalization of the endpoint protein levels. Together, these datasets provide a first view of the complexities associated with pharmacological rescue of learning in the Ts65Dn. Extending such studies to additional drugs and mouse models of DS will aid in identifying pharmacotherapies for effective clinical trials.

Protein profiles in Tc1 mice implicate novel pathway perturbations in the Down syndrome brain

Tc1 mouse model of Down syndrome (DS) is functionally trisomic for ∼120 human chromosome 21 (HSA21) classical protein-coding genes. Tc1 mice display features relevant to the DS phenotype, including abnormalities in learning and memory and synaptic plasticity. To determine the molecular basis for the phenotypic features, the levels of 90 phosphorylation-specific and phosphorylation-independent proteins were measured by Reverse Phase Protein Arrays in hippocampus and cortex, and 64 in cerebellum, of Tc1 mice and littermate controls. Abnormal levels of proteins involved in MAP kinase, mTOR, GSK3B and neuregulin signaling were identified in trisomic mice. In addition, altered correlations among the levels of N-methyl-D-aspartate (NMDA) receptor subunits and the HSA21 proteins amyloid beta (A4) precursor protein (APP) and TIAM1, and between immediate early gene (IEG) proteins and the HSA21 protein superoxide dismutase-1 (SOD1) were found in the hippocampus of Tc1 mice, suggesting altered stoichiometry among these sets of functionally interacting proteins. Protein abnormalities in Tc1 mice were compared with the results of a similar analysis of Ts65Dn mice, a DS mouse model that is trisomic for orthologs of 50 genes trisomic in the Tc1 plus an additional 38 HSA21 orthologs. While there are similarities, abnormalities unique to the Tc1 include increased levels of the S100B calcium-binding protein, mTOR proteins RAPTOR and P70S6, the AMP-kinase catalytic subunit AMPKA, the IEG proteins FBJ murine osteosarcoma viral oncogene homolog (CFOS) and activity-regulated cytoskeleton-associated protein (ARC), and the neuregulin 1 receptor ERBB4. These data identify novel perturbations, relevant to neurological function and to some seen in Alzheimers disease, that may occur in the DS brain, potentially contributing to phenotypic features and influencing drug responses.

Expression of trisomic proteins in Down syndrome model systems.

Down syndrome (DS) is the most common genetic aberration leading to intellectual disability. DS results from an extra copy of the long arm of human chromosome 21 (HSA21) and the increased expression of trisomic genes due to gene dosage. While expression in DS and DS models has been studied extensively at the RNA level, much less is known about expression of trisomic genes at the protein level. We have used quantitative Western blotting with antibodies to 20 proteins encoded by HSA21 to assess trisomic protein expression in lymphoblastoid cell lines (LCLs) from patients with DS and in brains from two mouse models of DS. These antibodies have recently become available and the 20 proteins largely have not been investigated previously for their potential contributions to the phenotypic features of DS. Twelve proteins had detectable expression in LCLs and three, CCT8, MX1 and PWP2, showed elevated levels in LCLs derived from patients with DS compared with controls. Antibodies against 15 proteins detected bands of appropriate sizes in lysates from mouse brain cortex. Genes for 12 of these proteins are trisomic in the Tc1 mouse model of DS, but only SIM2 and ZNF295 showed elevated expression in Tc1 cortex when compared with controls. Genes for eight of the 15 proteins are trisomic in the Ts65Dn mouse model of DS, but only ZNF294 was over expressed in cortex. Comparison of trisomic gene expression at the protein level with previous reports at the mRNA level showed many inconsistencies. These may be caused by natural inter-individual variability, differences in the age of mice analyzed, or post-transcriptional regulation of gene dosage effects. These antibodies provide resources for further investigation of the molecular basis of intellectual disability in DS.

Protein Profiles Associated With Context Fear Conditioning and Their Modulation by Memantine

Analysis of the molecular basis of learning and memory has revealed details of the roles played by many genes and the proteins they encode. Because most individual studies focus on a small number of proteins, many complexities of the relationships among proteins and their dynamic responses to stimulation are not known. We have used the technique of reverse phase protein arrays (RPPA) to assess the levels of more than 80 proteins/protein modifications in subcellular fractions from hippocampus and cortex of mice trained in Context Fear Conditioning (CFC). Proteins include components of signaling pathways, several encoded by immediate early genes or involved in apoptosis and inflammation, and subunits of glutamate receptors. At one hour after training, levels of more than half the proteins had changed in one or more fractions, among them multiple components of the Mitogen-activated protein kinase, MAPK, and Mechanistic Target of Rapamycin, MTOR, pathways, subunits of glutamate receptors, and the NOTCH pathway modulator, NUMB homolog (Drosophila). Levels of 37 proteins changed in the nuclear fraction of hippocampus alone. Abnormalities in levels of thirteen proteins analyzed have been reported in brains of patients with Alzheimers Disease. We therefore further investigated the protein profiles of mice treated with memantine, a drug approved for treatment of AD. In hippocampus, memantine alone induced many changes similar to those seen after CFC and altered the levels of seven proteins associated with Alzheimers Disease abnormalities. Lastly, to further explore the relevance of these datasets, we superimposed responses to CFC and memantine onto components of the long term potentiation pathway, a process subserving learning and memory formation. Fourteen components of the long term potentiation pathway and 26 proteins interacting with components responded to CFC and/or memantine. Together, these datasets provide a novel view of the diversity and complexity in protein responses and interactions following normal learning.

Abnormal Protein Profiles in Hippocampus of Mouse Models of Down Syndrome: Similarities with Alzheimers Disease

Down syndrome (DS) is caused by an extra copy of the long arm of human chromosome 21 (HSA21) and the increased expression, due to dosage, of HSA21 encoded genes. In addition to intellectual disability, all individuals with DS develop the neuropathology of Alzheimer’s Disease (AD) by age 30-40. The amyloid precursor protein gene, APP, that is mutated or duplicated in some familial AD (FAD), is encoded by HSA21, over expressed in DS, and a candidate for causing AD in DS. However, only half of those with DS will develop the AD-like dementia by age 50-60, suggesting that additional HSA21 genes may modulate the effects of APP triplication, and/or protect the DS brain from early onset progression to dementia in spite of neuropathology. In sporadic AD and mouse models of FAD, abnormal levels of a diverse set of proteins, including receptors, scaffold proteins, kinases, phosphatases and cytokines, have been documented, but nothing is known about their possible roles in AD in DS. Here, we compare expression of 26 AD-related proteins in hippocampus of four mouse models of DS, the Ts65Dn, Tc1, Dp (10)1Yey and Dp (17)1Yey, that together provided trisomy of partially overlapping subsets of all HSA21 genes or mouse orthologs. In the Dp(10)1Yey, that is trisomic for HSA21 orthologs mapping to mouse chromosome 10, twelve of 26 AD-related proteins were elevated, while in the Tc1, Dp(17)1Yey and the popular Ts65Dn, six, four and two differed from littermate controls. These data suggest that genes mapping to the HSA21 orthologous regions of mouse chromosomes 10 and 17 contribute to protein perturbations in the DS brain, and possibly AD in DS. Considering the different phenotypic features of the four DS mouse models further suggests that some protein abnormalities may be compensatory and protective for brain function and/or that learning and memory deficits may be age-dependent.

GM-CSF REVERSES MEMORY DEFICITS IN THE DP16 MOUSE MODEL OF DOWN SYNDROME

gliosis, and Ab plaque load. Basal glutamate (A) was measured prior to stimulus-evoked (B; 70 mM KCI, isotonic, pH 7.4) glutamate release (C). IHC analysis of CA1 VGLUT1 (E) and GFAP (F) density was determined as well as whole hippocampal Ap plaque pathology. All data represent mean 6 SEM and a two-way ANOVA with Holm-Sidak post hoc was used for statistical analyses. For (A-C) *p<0.05 C57BL/6J LFD (n1⁄41314) vs C57BL/6J HFD (n1⁄410-11), #p<0.05 AbPP/PS1 LFD (n1⁄411) vs AbPP/PS1 HFD (n1⁄411), xp<0.05 C57BL/6J LFD vs AbPP/PS1 LFD, zp<0.05 C57BL/6J HFD vs AbPP/PS1 HFD. For (D-F) *p<0.05 C57BL/ 6J LFD (n1⁄413) vs C57BL/6J HFD (n1⁄48); ##p<0.01, ###p<0.001 AbPP/ PS1 LFD (n1⁄47-10) vs AbPP/PS1 HFD (n1⁄411-12); xxp<0.01, xxxxp<0.0001 C57BL/6J LFD (n1⁄413-14) vs AbPP/PS1 LFD; zzzzp<0.0001 C57BL/6J HFD vs AbPP/PS1 HFD. Poster Presentations: Monday, July 23, 2018 P730

The GABAAα5-selective Modulator, RO4938581, Rescues Protein Anomalies in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS), trisomy of human chromosome 21 (Hsa21), is the most common genetic cause of intellectual disability (ID). There are no treatments for the cognitive deficits. The Ts65Dn is a partial trisomy mouse model of DS that shows learning and memory (LM) impairments and other abnormalities relevant to those seen in DS. Many drugs and small molecules have been shown to rescue the LM deficits, but little is known about the associated molecular responses. Here, patterns of protein expression are described in hippocampus of Ts65Dn and euploid littermate controls exposed to a battery of LM and behavior tests with and without chronic treatment with the GABAA receptor α5 subunit-selective negative allosteric modulator, RO4938581, that rescued LM deficits. Levels of 91 proteins/protein modifications, selected for relevance to LM and synaptic plasticity, were measured: 44 of 52 abnormalities present in vehicle-treated Ts65Dn were corrected by RO4938581. Superimposing protein data onto the molecular pathway defining long-term potentiation (LTP) shows that profiles are consistent with both abnormal LTP in vehicle-treated Ts65Dn and its observed rescue by RO4938581. Lastly, comparing these results with those from Ts65Dn treated, using a different protocol, with the NMDA receptor antagonist, memantine, that also rescues LM impairments, identifies common and divergent responses to the two drugs. Expansion of this approach to include additional drugs and DS models would aid in determining critical protein abnormalities and in identifying cocktails of drugs and/or new drug targets that would be effective in clinical trials for ID in DS.