Research focused on Down syndrome has increased in the last several years to advance understanding of the consequences of trisomy 21 (T21) on molecular and cellular processes and, ultimately, on ...individuals with Down syndrome. The Trisomy 21 Research Society (T21RS) is the premier scientific organization for researchers and clinicians studying Down syndrome. The Third International Conference of T21RS, held June 6–9, 2019, in Barcelona, Spain, brought together 429 scientists, families, and industry representatives to share the latest discoveries on underlying cellular and molecular mechanisms of T21, define cognitive and behavioral challenges and better understand comorbidities associated with Down syndrome, including Alzheimer’s disease and leukemia. Presentation of cutting-edge results in neuroscience, neurology, model systems, psychology, cancer, biomarkers and molecular and pharmacological therapeutic approaches demonstrate the compelling interest and continuing advancement in all aspects of understanding and ameliorating conditions associated with T21.
Abstract Mouse models of Down syndrome (DS) exhibit abnormal brain developmental and neurodegenerative changes similar to those seen in individuals with DS. Although DS mice have been well ...characterized cognitively and morphologically there are no prior reports utilizing diffusion MRI. In this study we investigated the ability of diffusional kurtosis imaging (DKI) to detect the progressive developmental and neurodegenerative changes in the Ts65Dn (TS) DS mouse model. TS mice displayed higher diffusional kurtosis (DK) in the frontal cortex (FC) compared to normal mice at 2 months of age. At 5 months of age, TS mice had lower radial kurtosis in the striatum (ST), which persisted in the 8-month-old mice. The TS mice exhibited lower DK metrics values in the dorsal hippocampus (HD) at all ages, and the group difference in this region was larger at 8-months. Regression analysis showed that normal mice had a significant age-related increase in DK metrics in FC, ST and HD. On the contrary, the TS mice lacked significant age-related increase in DK metrics in FC and ST. Although preliminary, these results demonstrate that DK metrics can detect TS brain developmental and neurodegenerative abnormalities.
Background
Calbindin‐D28k (CB) is a calcium‐binding/buffering protein that plays critical roles in preventing neuronal death as well as maintaining calcium homeostasis. Marked reductions in neuronal ...CB expression have been previously observed in individuals suffering from Alzheimer’s disease (AD) and recently observed in individuals with Down syndrome (DS) who develop early‐onset AD in the mid‐40s. The Ts65Dn mouse model of DS develops neurodegenerative patterns in the basal forebrain, the locus coeruleus and in the hippocampal perforant path. We hypothesized that neuronal expression of the mouse CB‐homologue, calbindin 1, may also be reduced in Ts65Dn mice and associated with cognitive deficits and neurodegeneration.
Method
Male Ts65Dn mice (n = 24) and euploid control mice (n = 26) were obtained from Jackson Laboratory (Bar Harbor, ME). Mouse cohorts were either 4‐5 months of age (pre‐neurodegeneration) or 8‐10 months of age (post‐neurodegeneration). A behavioral battery was administered to quantify spontaneous activity (SA), novel object recognition(NORT), and spatial memory (WRAM). We performed immunostaining and immunoblotting of the CB‐homologue (calbindin 1) in cortical areas and in the hippocampal formation. performed two‐way ANOVAs to test for differences between the groups (karyotype x treatment), followed by Tukey’s post hoc analyses. Correlations and linear regressions were performed between calbindin 1 and cognitive performance measures. All statistical tests were performed with GraphPad Prism 7.03 (GraphPad Software, La Jolla, CA).
Result
In the adult hippocampus and in cortical areas, calbindin 1 expression was observed in specific principal neurons and subsets of interneurons for all mice. Ts65Dn mice were found to have significant reductions (15‐30% ) of calbindin 1 at 4 months of age. However, loss of calbindin 1‐positive principal neurons in the CA1 was more prominent at 8‐10 months of age in Ts65Dn mice. Calbindin 1 expression in the hippocampus and parietal cortex (but not frontal cortex) significantly correlated with SA and WRAM. Linear regression confirmed the goodness of fit and non‐deviation from linearity for all correlations.
Conclusion
Early calbindin 1 depletion contributes to neuronal deficits and cognitive dysfunction in the Ts65Dn mouse model of DS mouse models. These results may be highly translatable to DS‐AD, where a similar CB phenotype also occurs.
Abstract
Background
Calbindin‐D28k (CB) is a calcium‐binding/buffering protein that plays critical roles in preventing neuronal death as well as maintaining calcium homeostasis. Marked reductions in ...neuronal CB expression have been previously observed in individuals suffering from Alzheimer’s disease (AD) and recently observed in individuals with Down syndrome (DS) who develop early‐onset AD in the mid‐40s. The Ts65Dn mouse model of DS develops neurodegenerative patterns in the basal forebrain, the locus coeruleus and in the hippocampal perforant path. We hypothesized that neuronal expression of the mouse CB‐homologue, calbindin 1, may also be reduced in Ts65Dn mice and associated with cognitive deficits and neurodegeneration.
Method
Male Ts65Dn mice (n = 24) and euploid control mice (n = 26) were obtained from Jackson Laboratory (Bar Harbor, ME). Mouse cohorts were either 4‐5 months of age (pre‐neurodegeneration) or 8‐10 months of age (post‐neurodegeneration). A behavioral battery was administered to quantify spontaneous activity (SA), novel object recognition(NORT), and spatial memory (WRAM). We performed immunostaining and immunoblotting of the CB‐homologue (calbindin 1) in cortical areas and in the hippocampal formation. performed two‐way ANOVAs to test for differences between the groups (karyotype x treatment), followed by Tukey’s post hoc analyses. Correlations and linear regressions were performed between calbindin 1 and cognitive performance measures. All statistical tests were performed with GraphPad Prism 7.03 (GraphPad Software, La Jolla, CA).
Result
In the adult hippocampus and in cortical areas, calbindin 1 expression was observed in specific principal neurons and subsets of interneurons for all mice. Ts65Dn mice were found to have significant reductions (15‐30% ) of calbindin 1 at 4 months of age. However, loss of calbindin 1‐positive principal neurons in the CA1 was more prominent at 8‐10 months of age in Ts65Dn mice. Calbindin 1 expression in the hippocampus and parietal cortex (but not frontal cortex) significantly correlated with SA and WRAM. Linear regression confirmed the goodness of fit and non‐deviation from linearity for all correlations.
Conclusion
Early calbindin 1 depletion contributes to neuronal deficits and cognitive dysfunction in the Ts65Dn mouse model of DS mouse models. These results may be highly translatable to DS‐AD, where a similar CB phenotype also occurs.
The ubiquitin–proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, ...muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate-specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing tandem ubiquitin binding entities technology, two-dimensional differential in gel electrophoresis, and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets.
Background
Brain pericytes maintain the integrity of the blood‐brain barrier (BBB) and facilitate the removal of amyloid β (Aβ) which is critical to healthy brain activity (Ethan et al., Brain ...Pathol., 24: 371‐386, 2014). Pericyte degeneration has been observed in brains from patients with Alzheimer’s disease (AD) and animal models (Jesse et al., Brain Pathol., 23: 303‐10, 2013; Wu et al., Aging (Albany NY), 11: 6120‐6133, 2019). Our previous data demonstrated that friend leukemia virus integration 1 (Fli‐1), an ETS transcription factor, regulates pericyte viability and vascular injury in murine sepsis (Li et al., J Infect Dis, 218: 1995‐2005, 2018); however, the role of Fli‐1 in AD remains unknown.
Methods
Fli‐1 levels and pericyte number were detected in postmortem brains from 21 patients with AD and 17 healthy controls, and in 5xFAD transgenic mice. Control or Fli‐1 antisense oligonucleotide Gapmers were injected into the hippocampus of 5xFAD mice at 3 and 4.5 months of age. Pericyte number, inflammatory mediators, Aβ deposition, BBB function and cognitive deficits were evaluated at 6 months of age. Human brain pericytes were transfected with control or Fli‐1 antisense Gapmers for 48 h and cultured with or without freshly aggregated Aβ40 for 7 consecutive days. Cell viability and apoptosis were detected.
Results
We demonstrated that Fli‐1 expression was increased in postmortem brains from a cohort of human AD donors and in 5xFAD mice, which corresponded with a decreased pericyte number, elevated inflammatory mediators, and increased Aβ accumulation as compared to cognitively normal individuals and WT mice, respectively. Antisense oligonucleotide Fli‐1 Gapmer administrated via intrahippocampal injection decelerated pericyte loss, reduced inflammatory responses, ameliorated cognitive deficits, improved BBB dysfunction, and decreased Aβ deposition in 5xFAD mice. Fli‐1 Gapmer‐mediated inhibition of Fli‐1 protected against Aβ accumulation induced human brain pericyte apoptosis in vitro.
Conclusion
These composite results indicate that Fli‐1 contributes to pericyte loss, inflammatory response, Aβ deposition, vascular dysfunction and cognitive decline, and suggest that suppression of Fli‐1 may represent a novel therapeutic strategy for AD.
Apolipoprotein E (ApoE) is a major lipid carrier protein. In humans, ApoE is expressed in three polymorphic isoforms, which are encoded by three different alleles APOE2, APOE3, and APOE4. In the ...brains of Alzheimer's disease (AD) patients, each one of these three allelic isoforms is found in several “isoelectric” protein isoforms (qPI), i.e. protein isoforms resulting from PTMs altering the net charge (q) of the polypeptide. AD is a complex disease in which multiple causes and several risk factors affect the onset and disease outcome. A major risk factor for AD is ApoE4; therefore, it is important to characterize the different ApoE qPIs. We have implemented a detergent‐based method for isolation and quantitation of protein isoforms, and we found differences in the solubility of protein isoforms depending on the type of solvent used. In this manuscript, we describe these methods and applied them to young human‐ApoE targeted replacement mice. Our results indicate that there are no significant differences in the hippocampus proteome of these mice as a function of the APOE genotype.
Apolipoprotein
E
(
A
po
E
) is a major lipid carrier protein. In humans,
A
po
E
is expressed in three polymorphic isoforms, which are encoded by three different alleles
APOE
2,
APOE
3, and
APOE
4. In ...the brains of
A
lzheimer's disease (
AD
) patients, each one of these three allelic isoforms is found in several “isoelectric” protein isoforms (
q
PI
), i.e. protein isoforms resulting from
PTM
s altering the net charge (
q
) of the polypeptide.
AD
is a complex disease in which multiple causes and several risk factors affect the onset and disease outcome. A major risk factor for
AD
is
A
po
E
4; therefore, it is important to characterize the different
A
po
E
q
PI
s. We have implemented a detergent‐based method for isolation and quantitation of protein isoforms, and we found differences in the solubility of protein isoforms depending on the type of solvent used. In this manuscript, we describe these methods and applied them to young human‐
A
po
E
targeted replacement mice. Our results indicate that there are no significant differences in the hippocampus proteome of these mice as a function of the
APOE
genotype.
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