It has been more than 10 years since it was first proposed that the neurodegeneration in Alzheimer9s disease (AD) may be caused by deposition of amyloid β-peptide (Aβ) in plaques in brain tissue. According to the amyloid hypothesis, accumulation of Aβ in the brain is the primary influence driving AD pathogenesis. The rest of the disease process, including formation of neurofibrillary tangles containing tau protein, is proposed to result from an imbalance between Aβ production and Aβ clearance.

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The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics

Thirteen families have been described with an autosomal dominantly inherited dementia named frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17)(1-9), historically termed Pick's disease(10) Most FTDP-17 cases show neuronal and/or glial inclusions that stain positively with antibodies raised against the microtubule-associated protein Tau, although the Tau pathology varies considerably in both its quantity (or severity) and characteristics(1-8,12) Previous studies have mapped the FTDP-17 locus to a 2-centimorgan region on chromosome 17q2111; the tau gene also lies within this region We have now sequenced tau in FTDP-17 families and identified three missense mutations (G272V, P301L and R406W) and three mutations in the 5' splice site of exon in The splice-site mutations all destabilize a potential stem-loop structure which is probably involved in regulating the alternative splicing of exon10 (ref 13) This causes more frequent usage of the 5' splice site and an increased proportion of tan transcripts that include exon 10 The increase in exon 10(+) messenger RNA will increase the proportion of Tau containing four microtubule-binding repeats, which is consistent with the neuropathology described in several families with FTDP-17 (refs 12, 14)

Association of missense and 5′-splice-site mutations in tau with the inherited dementia FTDP-17

A consensus panel from the United States and Europe was convened recently to update and revise the 1997 consensus guidelines for the neuropathologic evaluation of Alzheimer's disease (AD) and other diseases of brain that are common in the elderly. The new guidelines recognize the pre-clinical stage of AD, enhance the assessment of AD to include amyloid accumulation as well as neurofibrillary change and neuritic plaques, establish protocols for the neuropathologic assessment of Lewy body disease, vascular brain injury, hippocampal sclerosis, and TDP-43 inclusions, and recommend standard approaches for the workup of cases and their clinico-pathologic correlation.

/pdf/national-institute-on-aging-alzheimer-s-association-5crbry24zc.pdf

National Institute on Aging-Alzheimer's Association guidelines for the neuropathologic assessment of Alzheimer's disease

We present a practical guide for the implementation of recently revised National Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease (AD). Major revisions from previous consensus criteria are: (1) recognition that AD neuropathologic changes may occur in the apparent absence of cognitive impairment, (2) an “ABC” score for AD neuropathologic change that incorporates histopathologic assessments of amyloid β deposits (A), staging of neurofibrillary tangles (B), and scoring of neuritic plaques (C), and (3) more detailed approaches for assessing commonly co-morbid conditions such as Lewy body disease, vascular brain injury, hippocampal sclerosis, and TAR DNA binding protein (TDP)-43 immunoreactive inclusions. Recommendations also are made for the minimum sampling of brain, preferred staining methods with acceptable alternatives, reporting of results, and clinico-pathologic correlations.

/pdf/national-institute-on-aging-alzheimer-s-association-4honfkm34a.pdf

National Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease: a practical approach

Many neurodegenerative diseases, including Alzheimer's and Parkinson's and the transmissible spongiform encephalopathies (prion diseases), are characterized at autopsy by neuronal loss and protein aggregates that are typically fibrillar. A convergence of evidence strongly suggests that protein aggregation is neurotoxic and not a product of cell death. However, the identity of the neurotoxic aggregate and the mechanism by which it disables and eventually kills a neuron are unknown. Both biophysical studies aimed at elucidating the precise mechanism of in vitro aggregation and animal modeling studies support the emerging notion that an ordered prefibrillar oligomer, or protofibril, may be responsible for cell death and that the fibrillar form that is typically observed at autopsy may actually be neuroprotective. A subpopulation of protofibrils may function as pathogenic amyloid pores. An analogous mechanism may explain the neurotoxicity of the prion protein; recent data demonstrates that the disease-associated, infectious form of the prion protein differs from the neurotoxic species. This review focuses on recent experimental studies aimed at identification and characterization of the neurotoxic protein aggregates.

Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders.

We describe a novel variant of Alzheimer's disease (AD) in a Finnish pedigree with 17 affected individuals of both sexes in three generations. The disease is characterized by progressive dementia which is, in most cases, preceded by spastic paraparesis. Neuropathological investigations revealed numerous, distinct, large, round and eosinophilic plaques as well as neurofibrillary tangles and amyloid angiopathy throughout the cerebral cortex. The predominant plaques resembled cotton wool balls and were immunoreactive for Abeta but lacked a congophilic dense core or marked plaque-related neuritic pathology. Molecular genetic analysis revealed that the disease was caused by a deletion of exon 9 (delta9) of the presenilin 1 (PS1) gene from the mRNA: unlike previous examples of the delta9 variant, the deletion was not caused by a splice acceptor site mutation.

A variant of Alzheimer's disease with spastic paraparesis and unusual plaques due to deletion of exon 9 of presenilin 1

An Australian family with autosomal dominant presenile nonspecific dementia was recently described. The disease results in behavioral changes, usually disinhibition, followed by the onset of dementia accompanied occasionally by parkinsonism. Twenty-eight affected individuals were identified with an age of onset of 39 to 66 years (mean, 53 +/- 8.9 years). We mapped the disease locus to an approximately 26-cM region of chromosome 17q21-22 with a maximum two-point LOD score of 2.87. Affected individuals share a common haplotype between markers D17S783 and D17S808. This region of chromosome 17 contains the loci for several neurodegenerative diseases that lack distinctive pathological features, suggesting that these dementias, collectively referred to as frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), are caused by mutations in the same gene. The entire coding region of five genes, mapped to the FTDP-17 candidate region, were also sequenced. This analysis included the microtubule-associated protein tau that is the major component of the paired helical filaments observed in Alzheimer's disease. No pathogenic mutations were identified in either the tau gene or in any of the other genes analyzed.

Localization of frontotemporal dementia with parkinsonism in an Australian kindred to chromosome 17q21-22

Figure 1: A) Complementation mapping of fs(1)A13041. Boxes represent either the portion of the chromosome deleted or duplicated. For deficiencies, green indicates complementing deletions and black indicates non-complementing deletions. For duplications, green indicates rescuing fragments while black indicates non-rescuing fragments. Numbers indicate genomic coordinates in bases along the X chromosome. B) Genomic PCR of wildtype (WT) and sovA1304-1 flies. Primers were designed to amplify genomic DNA encoding the 5' UTR region of the sov-RA transcript. C) Cartoon of the sov locus. Dark blue represents the sov gene region with the left arrow representing the sov-RA transcriptional start site and right arrow representing the sov-RB/RC transcriptional start site. Green represents the CR43496 gene region with the arrow representing the transcriptional start site. Red box represents the deleted segment in sovA1304-1 flies. Small rectangles represent untranslated regions while large boxes represent translated regions. Numbers indicate genomic coordinates in bases along the X chromosome. Description X-linked female sterile screens in Drosophila have led to a tremendous increase in our understanding of the genetic control of oogenesis (Gans et al. 1975; Mohler 1977; Komitopoulou et al. 1983). However, many of the loci in these screens have not been mapped to a single gene and therefore remain a rich resource for further elucidating the genetic control of female fertility. fs(1)A13041 is one such allele that is germline dependent and results in a degenerative ovary phenotype (Gans et al. 1975; Khipple and King 1976; Mulligan 1981; Wieschaus et al. 1981; Mulligan and Rasch 1985; Lamnissou and Gelti-Douka 1985). We were interested in determining the mutation that leads to sterility in fs(1)A13041 females. Previous recombination mapping had placed fs(1)A13041 at 19±2 cM on the X chromosome (Gans, Audit, and Masson 1975; Khipple and King 1976). We confirmed the previous mapping interval by meiotically mapping fs(1)A13041 5/6/2020 Open Access to the right of crossveinless (12 cM) and to the left of singed (22 cM). We began complementation tests for female sterility with known deficiencies tiling the crossveinless and singed region and placed the lesion within a roughly 235 kb region (Figure 1A, non-complementing Df(1)BSC276, BSC285, BSC286, BSC297, BSC351, BSC535, and sov) (Parks et al. 2004; Cook et al. 2012). Two duplications within this narrow region rescued fs(1)A13041 sterility and thus further narrowed down the possible location of the causal mutation (Figure 1A, Dp(1;3)DC486 and Dp(1;3)DC026) (Venken et al. 2010). The mapping results were somewhat ambiguous within this narrow region (discussed below). However, the smallest noncomplementing deficiency, Df(1)sov, contains only the protein coding gene small ovary (sov) and non-coding RNA gene CR43496. We therefore decided to complementation test fs(1)A13041 with known alleles of sov. Flies homozygous for hypomorphic alleles of sov show a similar female sterility phenotype to flies bearing fs(1)A13041 while amorphic sov alleles are embryonic lethal (Wayne et al. 1995; Jankovics et al. 2018; Benner et al. 2019). We found that amorphic alleles sovEA42 and sovML150 failed to complement fs(1)A13041 female sterility while the hypomorphic sov2 complemented fs(1)A13041 sterility. Collectively this indicates that fs(1)A13041 is a sov allele (sovA1304-1). To determine the molecular lesion, we performed paired-end DNA sequencing on sovA1304-1 females. The sov locus contains three annotated transcripts; sov-RA has an annotated upstream transcriptional start site while sov-RB/RC are annotated to use a downstream transcriptional start site (Thurmond et al. 2019). Our sequencing data suggested that sovA1304-1 flies contained a deletion within the sov gene region that would delete a majority of the sov-RA 5′ UTR. Genomic PCR of this potential deletion confirmed the presence of a deletion in sovA1304-1 flies (Figure 1B). Sanger sequencing of the sovA1304-1 genomic PCR product showed that there was a 324 nucleotide deletion (chrX:6,756,3856,756,709) and a 10 nucleotide insertion (TCAACCTTCG) in the sov-RA 5′ UTR and would therefore remove most of the annotated 5′ UTR and donor splice site (Figure 1C). We are unsure why a duplication (Dp(1;3)DC026) and a deficiency (Df(1)BSC535) to the left of the sov region rescued and failed to complement sovA1304-1, respectively. We also found that the small duplication of just sov and CR43496 (Dp(1;3)sovtCH322-191E24) failed to rescue. We were not able to find any deleterious mutations or structural variants in our sequencing data to the left of sov that might indicate the presence of a second-site suppressor or long-range genomic interactions with the sov locus that are necessary for its proper expression. It is interesting that sovA1304-1 had not been previously mapped to sov since the Mohler and Gans X-linked female sterile collections had been previously complementation tested inter se (Perrimon et al. 1986). We found that one of the original Mohler alleles, sov2, complemented sovA1304-1sterility and is thus possible that the other two Mohler alleles, sov1 and sov3, behaved similarly, providing an explanation as to why sovA1304-1 was not previously recognized as belonging to the sov locus. It would be interesting to determine if the 5′ UTR deletion of the sov-RA transcript found in sovA1304-1 flies affects sov activity in other tissues of the body other than the ovary. There is no indication that sov-RA, or sov-RB/RC, is differentially expressed in the ovary or other adult tissues (Benner et al. 2019). Pole cell transplantation studies of sovA1304-1 indicated that defects are germline dependent (Wieschaus et al. 1981; Lamnissou and Gelti-Douka 1985), however, sov is an essential gene that has been shown to dominantly suppress position-effect variegation in tissues such as the eye (Jankovics et al. 2018; Benner et al. 2019). It is possible that the deletion solely affects sov-RA and that the Drosophila ovary is more sensitive to loss of sov-RA, or sov transcripts in general, in comparison to other tissues since sovA1304-1 females are viable but sterile. However, we have not directly measured the deletions effects on sov-RB/RC transcript levels, which might also be perturbed. The nature of the sovA1304-1 deletion therefore provides a unique mechanism to further elucidate the function of Sov at potentially both the transcript and regulatory level in Drosophila. Methods Request a detailed protocol Flies were cultured on ‘Fly Food A’ (LabExpress, Ann Arbor, MI) under standard laboratory conditions at 25°C. Genomic DNA was extracted from 30 homozygous fs(1)A13041 flies with a Qiagen DNeasy Blood and Tissue Kit (Hilden, Germany) according to the manufacturers insect protocol. DNA-sequencing libraries were made with Illumina Nextera DNA Library Prep Kit (San Diego, CA). 50 nucleotide paired-end sequencing was performed (Illumina HiSeq 2500, CASAVA base calling). Sequencing reads were mapped with Hisat2 to the FlyBase r6.25 genome and are available at the SRA (SRP238927) (Kim et al. 2015; Thurmond et al. 2019). Variant calling was completed with mpileup and bcftools from SAMtools within the X chromosome region 6625450-6860753 (Li et al. 2009; Li 2011) followed with variant annotation software snpEFF (Cingolani et al. 2012). For structural variant calling, we used BreakDancer software (Chen et al. 2009). Sanger sequencing was completed by Genewiz (Plainfield, NJ).

https://www.micropublication.org/pdf/micropub-biology-000246

fs(1)A13041 is a 5' UTR deletion of the essential gene small ovary in Drosophila.

Balancer chromosomes contain multiple inversions that work to suppress crossing over and prevent recovery of most recombinant chromosomes, allowing for alleles to be kept long-term without selection. These balancers are incredible tools, but some alleles within larger inverted segments are still lost to rare double crossover events between the balanced and balancer chromosomes. This study details a new methodology of producing balancer chromosomes using CRISPR/Cas9 gene editing technology to create new inversions within the largest segment of the common X chromosome balancer, FM7c. We were able to create a new X chromosome balancer, FM8, and anticipate that this process can be used to not only create other balancers in Drosophila melanogaster, but other model organisms as well.

Steve Kucera

Papers

A variant of Alzheimer's disease with spastic paraparesis and unusual plaques due to deletion of exon 9 of presenilin 1

Localization of frontotemporal dementia with parkinsonism in an Australian kindred to chromosome 17q21-22

fs(1)A13041 is a 5' UTR deletion of the essential gene small ovary in Drosophila.

Creation of a new Drosophila melanogaster X chromosome balancer, First Multiple 8 ( FM8 ), using the CRISPR/Cas9 gene-editing system