Micro-RNA profiles of pathology and resilience in posterior cingulate cortex of cognitively intact elders.

The posterior cingulate cortex (PCC) is a key hub of the default mode network underlying autobiographical memory retrieval, which falters early in the progression of Alzheimer's disease (AD). We recently performed RNA sequencing of post-mortem PCC tissue samples from 26 elderly Rush Religious Orders Study participants who came to autopsy with an ante-mortem diagnosis of no cognitive impairment but who collectively displayed a range of Braak I-IV neurofibrillary tangle stages. Notably, cognitively unimpaired subjects displaying high Braak stages may represent cognitive resilience to AD pathology. Transcriptomic data revealed elevated synaptic and ATP-related gene expression in Braak Stages III/IV compared with Stages I/II, suggesting these pathways may be related to PCC resilience. We also mined expression profiles for small non-coding micro-RNAs (miRNAs), which regulate mRNA stability and may represent an underexplored potential mechanism of resilience through the fine-tuning of gene expression within complex cellular networks. Twelve miRNAs were identified as differentially expressed between Braak Stages I/II and III/IV. However, the extent to which the levels of all identified miRNAs were associated with subject demographics, neuropsychological test performance and/or neuropathological diagnostic criteria within this cohort was not explored. Here, we report that a total of 667 miRNAs are significantly associated (rho > 0.38, P < 0.05) with subject variables. There were significant positive correlations between miRNA expression levels and age, perceptual orientation and perceptual speed. By contrast, higher miRNA levels correlated negatively with semantic and episodic memory. Higher expression of 15 miRNAs associated with lower Braak Stages I-II and 47 miRNAs were associated with higher Braak Stages III-IV, suggesting additional mechanistic influences of PCC miRNA expression with resilience. Pathway analysis showed enrichment for miRNAs operating in pathways related to lysine degradation and fatty acid synthesis and metabolism. Finally, we demonstrated that the 12 resilience-related miRNAs differentially expressed in Braak Stages I/II versus Braak Stages III/IV were predicted to regulate mRNAs related to amyloid processing, tau and inflammation. In summary, we demonstrate a dynamic state wherein differential PCC miRNA levels are associated with cognitive performance and post-mortem neuropathological AD diagnostic criteria in cognitively intact elders. We posit these relationships may inform miRNA transcriptional alterations within the PCC relevant to potential early protective (resilience) or pathogenic (pre-clinical or prodromal) responses to disease pathogenesis and thus may be therapeutic targets.

Transcriptomic profiling of early synucleinopathy in rats induced with preformed fibrils.

Examination of early phases of synucleinopathy when inclusions are present, but long before neurodegeneration occurs, is critical to both understanding disease progression and the development of disease modifying therapies. The rat alpha-synuclein (α-syn) preformed fibril (PFF) model induces synchronized synucleinopathy that recapitulates the pathological features of Parkinson's disease (PD) and can be used to study synucleinopathy progression. In this model, phosphorylated α-syn (pSyn) inclusion-containing neurons and reactive microglia (major histocompatibility complex-II immunoreactive) peak in the substantia nigra pars compacta (SNpc) months before appreciable neurodegeneration. However, it remains unclear which specific genes are driving these phenotypic changes. To identify transcriptional changes associated with early synucleinopathy, we used laser capture microdissection of the SNpc paired with RNA sequencing (RNASeq). Precision collection of the SNpc allowed for the assessment of differential transcript expression in the nigral dopamine neurons and proximal glia. Transcripts upregulated in early synucleinopathy were mainly associated with an immune response, whereas transcripts downregulated were associated with neurotransmission and the dopamine pathway. A subset of 29 transcripts associated with neurotransmission/vesicular release and the dopamine pathway were verified in a separate cohort of males and females to confirm reproducibility. Within this subset, fluorescent in situ hybridization (FISH) was used to localize decreases in the Syt1 and Slc6a3 transcripts to pSyn inclusion-containing neurons. Identification of transcriptional changes in early synucleinopathy provides insight into the molecular mechanisms driving neurodegeneration.

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

Co-expression network analysis of frontal cortex during the progression of Alzheimer's disease.

Mechanisms of Alzheimer's disease (AD) and its putative prodromal stage, amnestic mild cognitive impairment (aMCI), involve the dysregulation of multiple candidate molecular pathways that drive selective cellular vulnerability in cognitive brain regions. However, the spatiotemporal overlap of markers for pathway dysregulation in different brain regions and cell types presents a challenge for pinpointing causal versus epiphenomenal changes characterizing disease progression. To approach this problem, we performed Weighted Gene Co-expression Network Analysis and STRING interactome analysis of gene expression patterns quantified in frontal cortex samples (Brodmann area 10) from subjects who died with a clinical diagnosis of no cognitive impairment, aMCI, or mild/moderate AD. Frontal cortex was chosen due to the relatively protracted involvement of this region in AD, which might reveal pathways associated with disease onset. A co-expressed network correlating with clinical diagnosis was functionally associated with insulin signaling, with insulin (INS) being the most highly connected gene within the network. Co-expressed networks correlating with neuropathological diagnostic criteria (e.g., NIA-Reagan Likelihood of AD) were associated with platelet-endothelium-leucocyte cell adhesion pathways and hypoxia-oxidative stress. Dysregulation of these functional pathways may represent incipient alterations impacting disease progression and the clinical presentation of aMCI and AD.

MicroRNA-298 reduces levels of human amyloid-ß precursor protein (APP), ß-site APP-converting enzyme 1 (BACE1) and specific tau protein moieties.

Alzheimer's disease (AD) is the most common age-related form of dementia, associated with deposition of intracellular neuronal tangles consisting primarily of hyperphosphorylated microtubule-associated protein tau (p-tau) and extracellular plaques primarily comprising amyloid- ß (Aß) peptide. The p-tau tangle unit is a posttranslational modification of normal tau protein. Aß is a neurotoxic peptide excised from the amyloid-ß precursor protein (APP) by ß-site APP-cleaving enzyme 1 (BACE1) and the γ-secretase complex. MicroRNAs (miRNAs) are short, single-stranded RNAs that modulate protein expression as part of the RNA-induced silencing complex (RISC). We identified miR-298 as a repressor of APP, BACE1, and the two primary forms of Aß (Aß40 and Aß42) in a primary human cell culture model. Further, we discovered a novel effect of miR-298 on posttranslational levels of two specific tau moieties. Notably, miR-298 significantly reduced levels of ~55 and 50 kDa forms of the tau protein without significant alterations of total tau or other forms. In vivo overexpression of human miR-298 resulted in nonsignificant reduction of APP, BACE1, and tau in mice. Moreover, we identified two miR-298 SNPs associated with higher cerebrospinal fluid (CSF) p-tau and lower CSF Aß42 levels in a cohort of human AD patients. Finally, levels of miR-298 varied in postmortem human temporal lobe between AD patients and age-matched non-AD controls. Our results suggest that miR-298 may be a suitable target for AD therapy.

Dysfunctional neuroplasticity in newly arrived Middle Eastern refugees in the U.S.: Association with environmental exposures and mental health symptoms.

BACKGROUND: Psychological war trauma among displaced refugees is an established risk factor for mental health disorders, especially post-traumatic stress disorder (PTSD). Persons with trauma-induced disorders have heightened neuroplastic restructuring of limbic brain circuits (e.g., amygdala and hippocampus), which are critical factors in the pathophysiology of PTSD. Civilians in war are exposed to both psychological trauma and environmental hazards, such as metals. Little is known about the possible mental health impact from such environmental exposures, alone or in combination with trauma. It is of special interest to determine whether war exposures contribute to dysfunctional neuroplasticity; that is, an adverse outcome from sustained stress contributing to mental health disorders. The current study examined Middle Eastern refugees in the United States to determine the relationships among pre-displacement trauma and environmental exposures, brain derived neurotrophic growth factor (BDNF) and nerve growth factor (NGF)-two neurotrophins reported to mediate neuroplasticity responses to stress-related exposures-and mental health. METHODS: Middle Eastern refugees (n = 64; 33 men, 31 women) from Syria (n = 40) or Iraq (n = 24) were assessed 1 month after arrival to Michigan, US. Participants were interviewed in Arabic using a semi-structured survey to assess pre-displacement trauma and environmental exposure, PTSD, depression, anxiety, and self-rated mental health. Whole blood was collected, and concentrations of six heavy metals as well as BDNF and NGF levels were determined. Because these two neurotrophins have similar functions in neuroplasticity, we combined them to create a neuroplasticity index. Linear regression tested whether psychosocial trauma, environmental exposures and biomarkers were associated with mental health symptoms. FINDINGS: The neuroplasticity index was associated with PTSD (standardized beta, ß = 0.25, p < 0.05), depression (0.26, < 0.05) and anxiety (0.32, < 0.01) after controlling for pre-displacement trauma exposures. In addition, pre-displacement environmental exposure was associated with PTSD (0.28, < 0.05) and anxiety (0.32, < 0.05). Syrian refugees and female gender were associated with higher scores on depression (0.25, < 0.05; 0.30, < 0.05) and anxiety scales (0.35, < 0.01; 0.27, < 0.05), and worse on self-rated mental health (0.32, < 0.05; 0.34, < 0.05). In bivariate analysis, the neuroplasticity index was related to blood lead levels (r = 0.40; p < 0.01). CONCLUSIONS: The current study confirms the adverse effects of war trauma on mental health. Higher levels of biomarkers of neuroplasticity correlated with worse mental health and higher blood lead levels. Higher neurotrophin levels in refugees might indicate dysfunctional neuroplasticity with increased consolidation of adverse war memories in the limbic system. Such a process may contribute to psychiatric symptoms. Further research is needed to clarify the pathobiological mechanisms linking war trauma and environmental exposures to adverse mental health.

Locus Coeruleus Degeneration Induces Forebrain Vascular Pathology in a Transgenic Rat Model of Alzheimer's Disease.

Noradrenergic locus coeruleus (LC) neuron loss is a significant feature of mild cognitive impairment and Alzheimer's disease (AD). The LC is the primary source of norepinephrine in the forebrain, where it modulates attention and memory in vulnerable cognitive regions such as prefrontal cortex (PFC) and hippocampus. Furthermore, LC-mediated norepinephrine signaling is thought to play a role in blood-brain barrier (BBB) maintenance and neurovascular coupling, suggesting that LC degeneration may impact the high comorbidity of cerebrovascular disease and AD. However, the extent to which LC projection system degeneration influences vascular pathology is not fully understood. To address this question in vivo, we stereotactically lesioned LC projection neurons innervating the PFC of six-month-old Tg344-19 AD rats using the noradrenergic immunotoxin, dopamine-ß-hydroxylase IgG-saporin (DBH-sap), or an untargeted control IgG-saporin (IgG-sap). DBH-sap-lesioned animals performed significantly worse than IgG-sap animals on the Barnes maze task in measures of both spatial and working memory. DBH-sap-lesioned rats also displayed increased amyloid and inflammation pathology compared to IgG-sap controls. However, we also discovered prominent parenchymal albumin extravasation with DBH-sap lesions indicative of BBB breakdown. Moreover, microvessel wall-to-lumen ratios were increased in the PFC of DBH-sap compared to IgG-sap rats, suggesting that LC deafferentation results in vascular remodeling. Finally, we noted an early emergence of amyloid angiopathy in the DBH-sap-lesioned Tg344-19 AD rats. Taken together, these data indicate that LC projection system degeneration is a nexus lesion that compromises both vascular and neuronal function in cognitive brain areas during the prodromal stages of AD.

Peri-Infarct Upregulation of the Oxytocin Receptor in Vascular Dementia.

Vascular dementia (VaD) is cognitive decline linked to reduced cerebral blood perfusion, yet there are few therapeutic options to protect cognitive function following cerebrovascular accidents. The purpose of this study was to profile gene expression changes unique to VaD to identify and characterize disease relevant changes that could offer clues for future therapeutic direction. Microarray-based profiling and validation studies of postmortem frontal cortex samples from VaD, Alzheimer disease, and age-matched control subjects revealed that the oxytocin receptor (OXTR) was strongly and differentially upregulated in VaD. Further characterization in fixed tissue from the same cases showed that OXTR upregulation occurs de novo around and within microinfarcts in peri-infarct reactive astrocytes as well as within vascular profiles, likely on microvascular endothelial cells. These results indicate that increased OXTR expression in peri-infarct regions may be a specific response to microvascular insults. Given the established OXTR signaling cascades that elicit antioxidant, anti-inflammatory, and pro-angiogenic responses, the present findings suggest that de novo OXTR expression in the peri-infarct space is a tissue-protective response by astroglial and vascular cells in the wake of ischemic damage that could be exploited as a therapeutic option for the preservation of cognition following cerebrovascular insults.

Ezrin Expression is Increased During Disease Progression in a Tauopathy Mouse Model and Alzheimer's Disease.

BACKGROUND: The lack of diagnostic tools and disease-modifying treatments against Alzheimer's disease (AD) and related disorders, collectively known as tauopathies, has led to a socioeconomic burden of epidemic proportion. Proteomics approaches can be used to identify novel proteome changes that could help us understand the pathogenesis of tau-related pathological hallmarks and/or cellular stress responses associated with tauopathy. These studies, however, need to be conducted taking into consideration brain region specificity and stage of neurodegeneration in order to provide insights about the pathological role of the identified proteins. METHODS: We used a tauopathy mouse model (JNPL3) that expresses human tau bearing a P301L mutation and develops motor impairment, the severity of which correlates with the increased accumulation of pathological tau. Tissue was dissected from asymptomatic and severely motor impaired JNPL3 mice as well as non-transgenic littermate controls and subjected to two-dimensional gel electrophoresis. Differentially abundant protein spots were identified by tandem mass spectrometry. Postmortem mild cognitive impairment (MCI), AD and normal aging controls were used to validate the pathological significance of the identified protein. RESULTS: Ezrin was identified as a protein that is upregulated in tau-mediated neurodegeneration. We demonstrate that Ezrin protein abundance increased in JNPL3 mice preceded motor impairment and was sustained in severely motor impaired mice. Ezrin expression was also increased in the temporal cortex of MCI and AD patients. CONCLUSION: The results demonstrate that increased Ezrin protein abundance changes are associated with the early stages of neurodegeneration in tauopathy models and human disease. Understanding the role of Ezrin in tauopathies such as AD may provide new insights for targeting tau-mediated neurodegeneration.

Enhancing mitochondrial proteostasis reduces amyloid-ß proteotoxicity.

Alzheimer's disease is a common and devastating disease characterized by aggregation of the amyloid-ß peptide. However, we know relatively little about the underlying molecular mechanisms or how to treat patients with Alzheimer's disease. Here we provide bioinformatic and experimental evidence of a conserved mitochondrial stress response signature present in diseases involving amyloid-ß proteotoxicity in human, mouse and Caenorhabditis elegans that involves the mitochondrial unfolded protein response and mitophagy pathways. Using a worm model of amyloid-ß proteotoxicity, GMC101, we recapitulated mitochondrial features and confirmed that the induction of this mitochondrial stress response was essential for the maintenance of mitochondrial proteostasis and health. Notably, increasing mitochondrial proteostasis by pharmacologically and genetically targeting mitochondrial translation and mitophagy increases the fitness and lifespan of GMC101 worms and reduces amyloid aggregation in cells, worms and in transgenic mouse models of Alzheimer's disease. Our data support the relevance of enhancing mitochondrial proteostasis to delay amyloid-ß proteotoxic diseases, such as Alzheimer's disease.

Evidence for Mitochondrial UPR Gene Activation in Familial and Sporadic Alzheimer's Disease.

Mitochondrial perturbations such as oxidative stress, increased fission/fusion dysfunction, and mitophagy are consistent features of Alzheimer's disease (AD), yet the mechanisms that initiate these perturbations are unclear. One potential source for mitochondrial defects could be an imbalance in mitochondrial proteostasis. In this regard, studies indicate that a specialized mitochondrial unfolded protein response (mtUPR) is activated upon the aberrant accumulation of damaged or unfolded proteins in the mitochondrial matrix, resulting in the up-regulation of key genes involved in mitochondrial stabilization. To test whether mtUPR activation occurs in AD, we performed real-time quantitative PCR on postmortem frontal cortex samples from subjects classified as sporadic AD, familial AD linked to presenilin-1 mutations, or cognitively intact controls. Compared to controls, sporadic AD subjects exhibited a significant ~40-60% increase in expression levels of select genes activated by the mtUPR, including mitochondrial chaperones dnaja3, hspd1, and hspe1, mitochondrial proteases clpp and yme1l1, and txn2, a mitochondrial-specific oxidoreductase. Furthermore, levels of all six mtUPR genes were significantly up-regulated by ~70-90% in familial AD compared to controls, and these expression levels were significantly higher compared to sporadic AD. The increase in hspd1 (Hsp60) was validated by western blotting. These data support the concept that both sporadic and familial AD are characterized by mtUPR gene activation. Understanding the physiological consequences of this response may provide subcellular mechanistic clues to selective neuronal vulnerability or endogenous compensatory mechanisms during the progression of AD.

High correlations in gene expression between paired umbilical cord blood and neonatal blood of healthy newborns on Guthrie cards.

OBJECTIVE: To examine the correlation in genes expressed in paired umbilical cord blood (UCB) and newborn blood (NB). METHOD: Total mRNA and mRNA of three gene sets (inflammatory, hypoxia, and thyroidal response) was assessed using microarray in UCB and NB spotted on Guthrie cards from 7 mother/infant pairs. RESULTS: The average gene expression correlation between paired UCB and NB samples was 0.941 when all expressed genes were considered, and 0.949 for three selected gene sets. CONCLUSION: The high correlation of UCB and NB gene expression suggest that either source may be useful for examining gene expression in the perinatal period.

Partial deletion of the sulfate transporter SLC13A1 is associated with an osteochondrodysplasia in the Miniature Poodle breed.

A crippling dwarfism was first described in the Miniature Poodle in Great Britain in 1956. Here, we resolve the genetic basis of this recessively inherited disorder. A case-control analysis (8:8) of genotype data from 173 k SNPs revealed a single associated locus on CFA14 (P(raw) <10(-8)). All affected dogs were homozygous for an ancestral haplotype consistent with a founder effect and an identical-by-descent mutation. Systematic failure of nine, nearly contiguous SNPs, was observed solely in affected dogs, suggesting a deletion was the causal mutation. A 130-kb deletion was confirmed both by fluorescence in situ hybridization (FISH) analysis and by cloning the physical breakpoints. The mutation was perfectly associated in all cases and obligate heterozygotes. The deletion ablated all but the first exon of SLC13A1, a sodium/sulfate symporter responsible for regulating serum levels of inorganic sulfate. Our results corroborate earlier findings from an Slc13a1 mouse knockout, which resulted in hyposulfatemia and syndromic defects. Interestingly, the metabolic disorder in Miniature Poodles appears to share more clinical signs with a spectrum of human disorders caused by SLC26A2 than with the mouse Slc13a1 model. SLC26A2 is the primary sodium-independent sulfate transporter in cartilage and bone and is important for the sulfation of proteoglycans such as aggregan. We propose that disruption of SLC13A1 in the dog similarly causes undersulfation of proteoglycans in the extracellular matrix (ECM), which impacts the conversion of cartilage to bone. A co-dominant DNA test of the deletion was developed to enable breeders to avoid producing affected dogs and to selectively eliminate the mutation from the gene pool.

Copy number variations and primary open-angle glaucoma.

PURPOSE: This study sought to investigate the role of rare copy number variation (CNV) in age-related disorders of blindness, with a focus on primary open-angle glaucoma (POAG). Data are reported from a whole-genome copy number screen in a large cohort of 400 individuals with POAG and 500 age-matched glaucoma-free subjects. METHODS: DNA samples from patients and controls were tested for CNVs using a combination of two microarray platforms. The signal intensity data generated from these arrays were then analyzed with multiple CNV detection programs including CNAG version 2.0, PennCNV, and dChip. RESULTS: A total of 11 validated CNVs were identified as recurrent in the POAG set and absent in the age-matched control set. This set included CNVs on 5q23.1 (DMXL1, DTWD2), 20p12 (PAK7), 12q14 (C12orf56, XPOT, TBK1, and RASSF3), 12p13.33 (TULP3), and 10q34.21 (PAX2), among others. The CNVs presented here are exceedingly rare and are not found in the Database of Genomic Variants. Moreover, expression data from ocular tissue support the role of these CNV-implicated genes in vision-related processes. In addition, CNV locations of DMXL1 and PAK7 overlap previously identified linkage signals for glaucoma on 5p23.1 and 20p12, respectively. CONCLUSIONS: The data are consistent with the hypothesis that rare CNV plays a role in the development of POAG.

The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness.

The gene coding for centrosomal protein 290 (CEP290), a large multidomain protein, is the most frequently mutated gene underlying the non-syndromic blinding disorder Leber's congenital amaurosis (LCA). CEP290 has also been implicated in several cilia-related syndromic disorders including Meckel-Gruber syndrome, Joubert syndrome, Senor-Loken syndrome and Bardet-Biedl syndrome (BBS). In this study, we characterize the developmental and functional roles of cep290 in zebrafish. An antisense oligonucleotide [Morpholino (MO)], designed to generate an altered cep290 splice product that models the most common LCA mutation, was used for gene knockdown. We show that cep290 MO-injected embryos have reduced Kupffer's vesicle size and delays in melanosome transport, two phenotypes that are observed upon knockdown of bbs genes in zebrafish. Consistent with a role in cilia function, the cep290 MO-injected embryos exhibited a curved body axis. Patients with LCA caused by mutations in CEP290 have reduced visual perception, although they present with a fully laminated retina. Similarly, the histological examination of retinas from cep290 MO-injected zebrafish revealed no gross lamination defects, yet the embryos had a statistically significant reduction in visual function. Finally, we demonstrate that the vision impairment caused by the disruption of cep290 can be rescued by expressing only the N-terminal region of the human CEP290 protein. These data reveal that a specific region of the CEP290 protein is sufficient to restore visual function and this region may be a viable gene therapy target for LCA patients with mutations in CEP290.

Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome.

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly. We now describe serum-regulated upstream vesicular transport events leading to centrosomal Rab8 activation and ciliary membrane formation. Using live microscopy imaging, we show that upon serum withdrawal Rab8 is observed to assemble the ciliary membrane in ∼100 min. Rab8-dependent ciliary assembly is initiated by the relocalization of Rabin8 to Rab11-positive vesicles that are transported to the centrosome. After ciliogenesis, Rab8 ciliary transport is strongly reduced, and this reduction appears to be associated with decreased Rabin8 centrosomal accumulation. Rab11-GTP associates with the Rabin8 COOH-terminal region and is required for Rabin8 preciliary membrane trafficking to the centrosome and for ciliogenesis. Using zebrafish as a model organism, we show that Rabin8 and Rab11 are associated with the BBS pathway. Finally, using tandem affinity purification and mass spectrometry, we determined that the transport protein particle (TRAPP) II complex associates with the Rabin8 NH(2)-terminal domain and show that TRAPP II subunits colocalize with centrosomal Rabin8 and are required for Rabin8 preciliary targeting and ciliogenesis.

Genome-wide analysis of copy number variants in age-related macular degeneration.

Age-related macular degeneration (AMD) is a complex genetic disease, with many loci demonstrating appreciable attributable disease risk. Despite significant progress toward understanding the genetic and environmental etiology of AMD, identification of additional risk factors is necessary to fully appreciate and treat AMD pathology. In this study, we investigated copy number variants (CNVs) as potential AMD risk variants in a cohort of 400 AMD patients and 500 AMD-free controls ascertained at the University of Iowa. We used three publicly available copy number programs to analyze signal intensity data from Affymetrix GeneChip SNP Microarrays. CNVs were ranked based on prevalence in the disease cohort and absence from the control group; high interest CNVs were subsequently confirmed by qPCR. While we did not observe a single-locus "risk CNV" that could account for a major fraction of AMD, we identified several rare and overlapping CNVs containing or flanking compelling candidate genes such as NPHP1 and EFEMP1. These and other candidate genes highlighted by this study deserve further scrutiny as sources of genetic risk for AMD.

BBS6, BBS10, and BBS12 form a complex with CCT/TRiC family chaperonins and mediate BBSome assembly.

Bardet-Biedl syndrome (BBS) is a human genetic disorder resulting in obesity, retinal degeneration, polydactyly, and nephropathy. Recent studies indicate that trafficking defects to the ciliary membrane are involved in this syndrome. Here, we show that a novel complex composed of three chaperonin-like BBS proteins (BBS6, BBS10, and BBS12) and CCT/TRiC family chaperonins mediates BBSome assembly, which transports vesicles to the cilia. Chaperonin-like BBS proteins interact with a subset of BBSome subunits and promote their association with CCT chaperonins. CCT activity is essential for BBSome assembly, and knockdown of CCT chaperonins in zebrafish results in BBS phenotypes. Many disease-causing mutations found in BBS6, BBS10, and BBS12 disrupt interactions among these BBS proteins. Our data demonstrate that BBS6, BBS10, and BBS12 are necessary for BBSome assembly, and that impaired BBSome assembly contributes to the etiology of BBS phenotypes associated with the loss of function of these three BBS genes.

A BBSome subunit links ciliogenesis, microtubule stability, and acetylation.

Primary cilium dysfunction affects the development and homeostasis of many organs in Bardet-Biedl syndrome (BBS). We recently showed that seven highly conserved BBS proteins form a stable complex, the BBSome, that functions in membrane trafficking to and inside the primary cilium. We have now discovered a BBSome subunit that we named BBIP10. Similar to other BBSome subunits, BBIP10 localizes to the primary cilium, BBIP10 is present exclusively in ciliated organisms, and depletion of BBIP10 yields characteristic BBS phenotypes in zebrafish. Unexpectedly, BBIP10 is required for cytoplasmic microtubule polymerization and acetylation, two functions not shared with any other BBSome subunits. Strikingly, inhibition of the tubulin deacetylase HDAC6 restores microtubule acetylation in BBIP10-depleted cells, and BBIP10 physically interacts with HDAC6. BBSome-bound BBIP10 may therefore function to couple acetylation of axonemal microtubules and ciliary membrane growth.

Genetic interaction between Bardet-Biedl syndrome genes and implications for limb patterning.

Bardet-Biedl syndrome (BBS) is a pleiotropic, genetically heterogeneous disorder characterized by obesity, retinopathy, polydactyly, cognitive impairment, renal and cardiac anomalies, as well as hypertension and diabetes. Multiple genes are known to independently cause BBS. These genes do not appear to code for the same functional category of proteins; yet, mutation of each results in a similar phenotype. Gene knockdown of different BBS genes in zebrafish shows strikingly overlapping phenotypes including defective melanosome transport and disruption of the ciliated Kupffer's vesicle. Here, we demonstrate that individual knockdown of bbs1 and bbs3 results in the same prototypical phenotypes as reported previously for other BBS genes. We utilize the zebrafish system to comprehensively determine whether simultaneous pair-wise knockdown of BBS genes reveals genetic interactions between BBS genes. Using this approach, we demonstrate eight genetic interactions between a subset of BBS genes. The synergistic relationships between distinct combinations are not due to functional redundancy but indicate specific interactions within a multi-subunit BBS complex. In addition, we utilize the zebrafish model system to investigate limb development. Human polydactyly is a cardinal feature of BBS not reproduced in BBS-mouse models. We evaluated zebrafish fin bud patterning and observed altered Sonic hedgehog (shh) expression and subsequent changes to fin skeletal elements. The SHH fin bud phenotype was also used to confirm specific genetic interactions between BBS genes. This study reveals an in vivo requirement for BBS function in limb bud patterning. Our results provide important new insights into the mechanism and biological significance of BBS.