Peter H. Frederikse

Amyloid-β and tau pathology of Alzheimer's disease induced by diabetes in a rabbit animal model.

Alzheimers disease (AD) is the major age-dependent disease of the brain, but what instigates late-onset AD is not yet clear. Epidemiological, animal model, and cell biology findings suggest links between AD and diabetes. Although AD pathology is accelerated by diabetes in mice engineered to accumulate human-sequence amyloid-β (Aβ) peptides, they do not adequately model non-inherited AD. We investigated AD-type pathology induced solely by diabetes in genetically unmodified rabbits which generate human-sequence Aβ peptides. After 15 weeks, alloxan-treated diabetic rabbits with expected high blood glucose showed ~5-fold increase in Aβ40/Aβ42 in cortex and hippocampus, and significantly, generated Aβ-derived assemblies found in human AD. Deposits of these putative pathogenic toxins were detected by Aβ/Aβ oligomer antibodies in brain parenchyma and surrounding vasculature, also co-localizing with markedly elevated levels of RAGE. Soluble brain extracts showed diabetes-induced buildup of Aβ oligomers on dot-blots. Phospho-tau also was clearly elevated, overlapping with βIII-tubulin along neuronal tracts. Indications of retina involvement in AD led to examination of AD-type pathology in diabetic retinas and showed Aβ accumulation in ganglion and inner nuclear cell layers using Aβ/oligomer antibodies, and RAGE again was elevated. Our study identifies emergence of AD pathology in brain and retina as a major consequence of diabetes; implicating dysfunctional insulin signaling in late-onset AD, and a potential relationship between Aβ-derived neurotoxins and retinal degeneration in aging and diabetes, as well as AD. AD-type pathology demonstrated in genetically unmodified rabbits calls attention to the considerable potential of the model for investigation of AD pathogenesis, diagnostics, and therapeutics.

Fragile X Syndrome FMRP Co-localizes with Regulatory Targets PSD-95, GABA Receptors, CaMKIIα, and mGluR5 at Fiber Cell Membranes in the Eye Lens

Fmr1 and FMRP underlie Fragile X Syndrome (FXS) and are linked with related autism spectrum disorders (ASD). Fmr1 also has an essential role in eye and lens development. Lenses express FMRP along with γ-aminobutyric acid (GABA) receptors (GABARs), post-synaptic density protein 95 (PSD-95), Tyr-phosphatase STEP, CaMKIIα and Alzheimer’s disease Aβ precursor protein, which are verified targets of FMRP regulation in neurons and outline major topics in FXS/ASD research. PSD-95 as well as CaMKIIα transcripts undergo polypryimidine tract binding protein dependent alternative splicing in lens, consistent with PSD-95 translation in lens. At least 13 GABAR subunits and GAD25/65/67 GABA metabolism enzymes are expressed in lenses beginning in embryonic development, matching neural development. Interestingly, GABAergic drugs (e.g. baclofen) studied as FXS/ASD therapeutics are shown to resolve developmental vision defects in experimental myopia. Here, we demonstrated that FMRP co-localizes at fiber cell membranes with PSD-95, GABAAδ, GABAAβ3, GABBR1, STEP, CaMKIIα, and mGluR5 in young adult lenses. GAD65 and GABA detection was greatest at the peri-nuclear lens region where fiber cell terminal differentiation occurs. These findings add to an extensive list of detailed parallels between fiber cell and neuron morphology and their lateral membrane spine/protrusions, also reflected in the shared expression of genes involved in the morphogenesis and function of these membrane structures, and shared use of associated regulatory mechanisms first described as distinguishing the neuronal phenotype. Future studies can determine if GABA levels currently studied as a FXS/ASD biomarker in the brain, and generated by GAD25/65/67 in a comparable cell environment in the lens, may be similarly responsive to Fmr1 mutation in lens. The present demonstration of FMRP and key regulatory targets in the lens identifies a potential for the lens to provide a new research venue, in the same individual, to inform about Fmr1/FMRP pathobiology in brain as well as lens.

GluA2 AMPA glutamate receptor subunit exhibits codon 607 Q/R RNA editing in the lens.

Regulated GluA2 AMPA receptor subunit expression, RNA editing, and membrane localization are fundamental determinants of neuronal Ca(2+) influx, and underlie basic functions such as memory and the primary brain disorder epilepsy. Consistent with this, AMPARs, and specifically GluA2, are targets of common antiepileptic drugs (AEDs) and antidepressants. Recently, epidemiological associations between epilepsy and increased cataract prevalence were found comparable to cataract links with diabetes and smoking. Similarly, use of AEDs and several antidepressants also showed links with increased cataract. Here, we demonstrated GluA2 in lenses, consistent with REST/NRSF and REST4 we described previously in lenses, as well as GluA1 and ADAR2 in the lens. Surprisingly, we found predominant neuron-like Q/R editing of GluA2 RNAs also occurs in the lens and evidence of lens GluA2 phosphorylation and STEP phosphatases linked with GluA2 membrane localization in neurons. This study is among the first to show GluA2 expression and predominant Q/R RNA editing in a non-neural cell. Our results suggest GluA2 AMPARs have related roles in lens physiology and disease processes, and provide evidence these anticonvulsant and antidepressant drug targets also occur in the lens.

miR-124, miR-125b, let-7 and Vesicle Transport Proteins in Squid Lenses in L. pealei

Purpose: Studies over the past several decades identified parallels between neuron and lens fiber cell morphology, development, and physiology. Consistent with this, mammalian lens fiber cells were shown to express a substantial complement of genes that cluster with respect to synaptic vesicle transport and exocytosis. Expression of these genes in these two cell types also appears consistent with similarities described between lens fiber cell lateral protrusions and neuronal dendrites. Recently, we showed vertebrate neurons and lens fiber cells share expression of a core set of factors that form an interlocking regulatory network which has a fundamental role in determining neural cell identity. These included the REST/NRSF transcription factor, neural RNA binding proteins and miR-124. In addition, we identified miR-125 and let-7 in mammalian lenses that have been shown to regulate dendrite formation in neurons. The present study examined expression of miR-124, miR-125, and let-7 as well as genes involved in vesicle transport in lens in the squid Loligo (also referred to as Doryteuthis) pealei. Methods: Northern blot, RT-PCR, immunoblots, and in situ detection were used to analyze expression in squid and vertebrate tissues. Results: The present study provided evidence that miR-124, miR-125, let-7 and vesicle transport-related proteins are produced in squid lenses. Consistent with these mRNAs and miRNAs in squid lenses, and polyribosomes shown by others, we detected substantial levels of tRNA and rRNA in anuclear squid lenses which do not produce an epithelial cell layer that would be analogous to vertebrate lenses. Conclusions: Our study provided evidence that miR-124, miR-125, and let-7, as well as proteins involved in vesicle transport linked with synaptic and cargo vesicle transport in vertebrates are also expressed in squid lenses.

Increased expression and local accumulation of the Prion Protein, Alzheimer Aβ peptides, superoxide dismutase 1, and Nitric oxide synthases 1 & 2 in muscle in a rabbit model of diabetes

BackgroundMuscle disease associated with different etiologies has been shown to produce localized accumulations of amyloid and oxidative stress-related proteins that are more commonly associated with neurodegeneration in the brain. In this study we examined changes in muscle tissue in a classic model of diabetes and hyperglycemia in rabbits to determine if similar dysregulation of Alzheimer Aβ peptides, the prion protein (PrP), and superoxide dismutase 1 (SOD1), as well as nitric oxide synthases is produced in muscle in diabetic animals. This wild-type rabbit model includes systemic physiological expression of human-like Alzheimer precursor proteins and Aβ peptides that are considered key in Alzheimer protein studies.ResultsDiabetes was produced in rabbits by injection of the toxic glucose analogue alloxan, which selectively enters pancreatic beta cells and irreversibly decreases insulin production, similar to streptozotocin. Quadriceps muscle from rabbits 16 wks after onset of diabetes and hyperglycemia were analyzed with biochemical and in situ methods. Immunoblots of whole muscle protein samples demonstrated increased PrP, SOD1, as well as neuronal and inducible Nitric oxide synthases (NOS1 and NOS2) in diabetic muscle. In contrast, we detected little change in Alzheimer Aβ precursor protein expression, or BACE1 and Presenilin 1 levels. However, Aβ peptides measured by ELISA increased several fold in diabetic muscle, suggesting a key role for Aβ cleavage in muscle similar to Alzheimer neurodegeneration in this diabetes model. Histological changes in diabetic muscle included localized accumulations of PrP, Aβ, NOS1 and 2, and SOD1, and evidence of increased central nuclei and cell infiltration.ConclusionsThe present study provides evidence that several classic amyloid and oxidative stress-related disease proteins coordinately increase in overall expression and form localized accumulations in diabetic muscle. The present study highlights the capacity of this wild-type animal model to produce an array of hallmark pathological features that have also been described in other muscle diseases.

Lens GABA receptors are a target of GABA-related agonists that mitigate experimental myopia

Coordinated growth of eye tissues is required to achieve visual acuity. However, visual experience also guides this process. Experimental myopia can be produced by altering light entering the eye, but also by changing light/dark regimens. Drug discovery studies demonstrated that γ-aminobutyric acid (GABA)-related agonists (e.g., baclofen) will mitigate experimental myopia, and are also drugs studied for their capacity to affect neurodevelopmental disorders that include Fragile X Syndrome and related autism spectrum disorders. GABA receptors thought to mediate these responses in the eye have been studied in the neural retina as well as the cornea and sclera which are both innervated tissues. In addition to neurons, lenses express GAD25/65/67 GABA metabolic enzymes and at least 13 GABA receptor subunits with developmental expression profiles that match neural development. Evidence that lens GABA receptors are expressed in a cell environment comparable to neurons is seen in the lens expression of AMPA and NMDA glutamate receptors together with an unexpectedly comprehensive array of associated signaling proteins that include post-synaptic-density 95 (PSD95), calcium calmodulin kinase IIα (CaMKIIα), Fragile X Syndrome mental retardation protein (FMRP), ephrin receptors, Ca(V)1.2, 1.3 channels, cyclin-dependent kinase 5 (Cdk5), and neuronal C-src among others. Moreover, lens cells share fundamental molecular regulatory mechanisms that integrate the regulation and function of these genes at the DNA, RNA, and protein levels in neurons. GABA has trophic, growth promoting effects early in neuron development and later assumes its classic inhibitory role in the adult neural system. We hypothesize that the extensive parallels between GABA and glutamate receptor biology in lens and brain identifies the lens as a site of GABA agonist drug action affecting experimental myopia, acting through lens GABA receptors to similarly affect growth in both elongated cell types.

NMDA Glutamate Receptor NR1, NR2A and NR2B Expression and NR2B Tyr-1472 Phosphorylation in the Lens

Detailed parallels described between lens fiber cell and neuron morphology, sub-cellular structure, and molecular biology include striking similarities in the ultrastructure of their vesicle transport machinery and the membrane protrusions that occur along the lateral surfaces of both cell types. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate receptor (NMDA) glutamate receptors (AMPARs/NMDARs) are the predominant receptors in neurons. These receptors have fundamental roles in neuron morphogenesis as well as neuron physiology and dynamic cell signaling, and specifically at dendritic spines. As a result, AMPAR and NMDAR dysregulation underlies several primary neural disorders that have also shown epidemiological associations with cataract. Previously, we demonstrated AMPAR GluA1 and REST (RE-1 silencing transcription factor)-regulated GluA2 subunits are expressed in the lens, and showed C-terminal phospho-tyrosine-GluA2, and striatal-enriched tyrosine phosphatase (STEP), as well as GluA2 Q/R RNA editing in lenses similar to neurons. Here, we demonstrated that REST-regulated NMDAR NR1, NR2A, and NR2B are also expressed in lenses and localize predominantly in fiber cell membranes, consistent with REST transcription factors, as well as miR-124 and other REST gene targets identified in the lens. We also showed NR2B Tyr-1472 phosphorylation occurs in lens. These p-Tyr-GluA2 and p-Tyr-NR2B phosphorylation events are linked with membrane insertion regulated by STEP. We next determined that NR1 transcripts that include exon 5 are produced in lens consistent with Fox-1 RNA binding protein isoforms linked with this alternative splicing event, and shown to be expressed in lens as well as brain. These findings provide further evidence that fundamental neuronal morphogenetic programs, and hallmark neuronal gene expression and modes of regulation, are shared with elongated fiber cells of the lens.

“Moonlighting” GAPDH Protein Localizes with AMPA Receptor GluA2 and L1 Axonal Cell Adhesion Molecule at Fiber Cell Borders in the Lens

Abstract Purpose: The canonical role of glyceraldehyde phosphate dehydrogenase (GAPDH) is as an enzyme in glycolysis. GAPDH is also a principal “moonlighting” protein with additional roles at diverse sites in a variety of cells. Surface GAPDH on mammalian, yeast, and bacterial cells acts as a receptor and also mediates cell contacts. In neurons, extracellular GAPDH localizes at synapses. Two GAPDH binding partners at synapses are α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor (AMPA) GluA2 subunit at dendritic spines and L1 cell adhesion molecule at pre-synaptic membranes, and both proteins are also expressed in lenses. Fiber cell membrane protrusions and dendritic spines have similar size, shape, and spacing, contain F-actin, and express clathrin/AP-2 Adaptor at their surfaces linked with Tyr-phosphatase STEP-regulated endocytosis of AMPA/GluA2 receptors. AMPA receptors work with NMDA (N-methyl-d-aspartate) and GABA (γ-aminobutyric acid) receptors, calcium calmodulin kinase II (CaMKIIα), channel proteins, STEP, and ephrin receptors, which are also expressed in lenses. In neurons, coordinate AMPA/GluA2 receptor endocytosis with GAPDH is linked with disease. GAPDH was previously characterized as a fiber cell membrane protein and shown to decrease substantially in interior fiber cells in human age-related cataract. Here, we examined GAPDH spatial expression in healthy lenses in two vertebrate species. Methods: In situ methods were used to examine GAPDH expression in lenses of healthy young adult rabbits and chickens. Immunoblots were used to detect L1 in lenses. Results: The present study demonstrated that GAPDH is present at fiber cell borders in adult rabbit and chicken lenses with evidence of focal concentrations along the fiber cell perimeter, and overlapped with detection of p-Tyr-GluA2, L1, STEP, actin and clathrin. We observed that L1-140 kDa was the prominent form in lens. Conclusions: Our findings indicate investigations into GAPDH “moonlighting” activities similar to its role in cell–cell interactions at neuron surfaces are warranted in the lens.

Lens Biology is a Dimension of Neurobiology

There is a second cell type in your body that expresses scores of the most intensively studied genes in neuroscience and exclusively shares critical interdependent modes of molecular regulation that include a network first described as responsible for the basic bifurcation of neuronal from non-neuronal gene expression in vertebrates. Neurons and lens cells are among the most ancient animal cell types, yet neurons have an exclusive status also attributed to roles underlying sensation, movement, and cognition. However, this status is challenged by cells in the lens of the eye. The extent and detail of internally consistent parallels with neuron biology now catalogued in their second native cell type in the lens provide a detailed model of interdependent neuron gene expression in lens development and non-neuronal role in vision. These comprehensive parallels identify the lens as a dimension of neurobiology and a fundamental new perspective on neurodevelopment and its disorders. Finally, this understanding identifies that hallmark neuronal gene expression and key modes of associated molecular regulation evolved in tandem in the lens.

Spectrometry Detection of Alzheimer Biomarkers in AD in Diabetes

between the individual hypometabolism maps and the AD-like map, was calculated as the sum of the product of z-scores from each cerebral voxel from these two maps. HCI scores were compared between NC, HT and HM groups. Results: While the HM, HT, and NC groups did not differ significantly in their age, gender, educational level, or neuropsychological test scores, the three groups differed significantly in their HCI’s (24.38 6 4.84, 23.58 6 3.59, and 21.80 6 4.09, respectively; ANOVA, p 1⁄4 0.004). Additionally, there was a significant association between HCI’s and APOE e4 gene dose (linear trend, p 1⁄4 0.001). Conclusions: The AD-related HCI in cognitively normal late-middle-aged people is associated with three levels of genetic risk for late-onset AD. This index score offers promise in the pre-symptomatic detection and tracking of AD-associated hypometabolism, assessment of genetic and non-genetic risk factors, and evaluation of promising AD-modifying treatments in pre-symptomatic individuals.