Ida E. Holm

Nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration: an update

One year ago, in this journal, we published a recommended nomenclature for the neuropathologic subtypes of frontotemporal lobar degeneration (FTLD) [7]. A major impetus behind this was to resolve the confusion that had arisen around the use of the term “FTLD with ubiquitinated inclusions” (FTLD-U), following the discovery that the molecular pathology of these cases was heterogeneous, with most, but not all, being characterized by pathological TDP-43 [6, 11]. In addition, a system of nosology was introduced that grouped the FTLD subtypes into broad categories, based on the molecular defect that is most characteristic, according to current evidence. This system provided a concise and consistent terminology that has now been widely adopted in the literature. Another anticipated advantage was the ability to readily accommodate new discoveries. At the time, we did not anticipate how quickly this attribute would be put to use.

Hypothesis on the Dual Origin of the Mammalian Subplate

The development of the mammalian neocortex relies heavily on subplate. The proportion of this cell population varies considerably in different mammalian species. Subplate is almost undetectable in marsupials, forms a thin, but distinct layer in mouse and rat, a larger layer in carnivores and big-brained mammals as pig, and a highly developed embryonic structure in human and non-human primates. The evolutionary origin of subplate neurons is the subject of current debate. Some hypothesize that subplate represents the ancestral cortex of sauropsids, while others consider it to be an increasingly complex phylogenetic novelty of the mammalian neocortex. Here we review recent work on expression of several genes that were originally identified in rodent as highly and differentially expressed in subplate. We relate these observations to cellular morphology, birthdating, and hodology in the dorsal cortex/dorsal pallium of several amniote species. Based on this reviewed evidence we argue for a third hypothesis according to which subplate contains both ancestral and newly derived cell populations. We propose that the mammalian subplate originally derived from a phylogenetically ancient structure in the dorsal pallium of stem amniotes, but subsequently expanded with additional cell populations in the synapsid lineage to support an increasingly complex cortical plate development. Further understanding of the detailed molecular taxonomy, somatodendritic morphology, and connectivity of subplate in a comparative context should contribute to the identification of the ancestral and newly evolved populations of subplate neurons.

Genetically Modified Pig Models for Neurodegenerative Disorders

Increasing incidence of neurodegenerative disorders such as Alzheimers disease and Parkinsons disease has become one of the most challenging health issues in ageing humans. One approach to combat this is to generate genetically modified animal models of neurodegenerative disorders for studying pathogenesis, prognosis, diagnosis, treatment, and prevention. Owing to the genetic, anatomic, physiologic, pathologic, and neurologic similarities between pigs and humans, genetically modified pig models of neurodegenerative disorders have been attractive large animal models to bridge the gap of preclinical investigations between rodents and humans. In this review, we provide a neuroanatomical overview in pigs and summarize and discuss the generation of genetically modified pig models of neurodegenerative disorders including Alzheimers diseases, Huntingtons disease, Parkinsons disease, amyotrophic lateral sclerosis, spinal muscular atrophy, and ataxia–telangiectasia. We also highlight how non‐invasive bioimaging technologies such as positron emission tomography (PET), computer tomography (CT), and magnetic resonance imaging (MRI), and behavioural testing have been applied to characterize neurodegenerative pig models. We further propose a multiplex genome editing and preterm recloning (MAP) approach by using the rapid growth of the ground‐breaking precision genome editing technology CRISPR/Cas9 and somatic cell nuclear transfer (SCNT). With this approach, we hope to shorten the temporal requirement in generating multiple transgenic pigs, increase the survival rate of founder pigs, and generate genetically modified pigs that will more closely resemble the disease‐causing mutations and recapitulate pathological features of human conditions. Copyright

Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT)

Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using minicircles in RMCE in fibroblasts with all four acceptor loci and followed by SCNT, we produced piglets with a single copy of a transgene incorporated into one of the transcriptionally active acceptor loci. The transgene, consisting of a cDNA of the Alzheimer’s disease-causing gene PSEN1M146I driven by an enhanced human UbiC promoter, had an expression profile in various tissues similar to that of the GFP marker gene. The results show that RMCE can be done in a pre-selected transcriptionally active acceptor locus for targeted transgenesis in pigs.

Mitochondria and the Lectin Pathway of Complement

Background: Mitochondria are remnants of a eubacterial endosymbiont and may elicit untoward inflammation. Results: MBL, L-ficolin, and M-ficolin recognize mitochondria. MBL binding activates complement. Upon mitochondrial challenge, C3 is consumed in vivo in the absence of overt inflammation. Conclusion: The lectin pathway may be involved in homeostatic clearance of mitochondria. Significance: This is the first indication of lectin pathway involvement in mitochondrial immune handling. Mitochondria, the powerhouses of our cells, are remnants of a eubacterial endosymbiont. Notwithstanding the evolutionary time that has passed since the initial endosymbiotic event, mitochondria have retained many hallmarks of their eubacterial origin. Recent studies have indicated that during perturbations of normal homeostasis, such as following acute trauma leading to massive necrosis and release of mitochondria, the immune system might mistake symbiont for enemy and initiate an inappropriate immune response. The innate immune system is the first line of defense against invading microbial pathogens, and as such is the primary suspect in the recognition of mitochondria-derived danger-associated molecular patterns and initiation of an aberrant response. Conversely, innate immune mechanisms are also central to noninflammatory clearance of innocuous agents. Here we investigated the role of a central humoral component of innate immunity, the lectin pathway of complement, in recognition of mitochondria in vitro and in vivo. We found that the soluble pattern recognition molecules, mannan-binding lectin (MBL), L-ficolin, and M-ficolin, were able to recognize mitochondria. Furthermore, MBL in complex with MBL-associated serine protease 2 (MASP-2) was able to activate the lectin pathway and deposit C4 onto mitochondria, suggesting that these molecules are involved either in homeostatic clearance of mitochondria or in induction of untoward inflammatory reactions. We found that following mitochondrial challenge, C3 was consumed in vivo in the absence of overt inflammation, indicating a potential role of complement in noninflammatory clearance of mitochondria. Thus, we report here the first indication of involvement of the lectin pathway in mitochondrial immune handling.

Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease.

Early microgliosis precedes neuronal loss and behavioural impairment in mice with a frontotemporal dementia-causing CHMP2B mutation

Abstract Frontotemporal dementia (FTD)‐causing mutations in the CHMP2B gene lead to the generation of mutant C‐terminally truncated CHMP2B. We report that transgenic mice expressing endogenous levels of mutant CHMP2B developed late‐onset brain volume loss associated with frank neuronal loss and FTD‐like changes in social behaviour. These data are the first to show neurodegeneration in mice expressing mutant CHMP2B and indicate that our mouse model is able to recapitulate neurodegenerative changes observed in FTD. Neuroinflammation has been increasingly implicated in neurodegeneration, including FTD. Therefore, we investigated neuroinflammation in our CHMP2B mutant mice. We observed very early microglial proliferation that develops into a clear pro‐inflammatory phenotype at late stages. Importantly, we also observed a similar inflammatory profile in CHMP2B patient frontal cortex. Aberrant microglial function has also been implicated in FTD caused by GRN, MAPT and C9orf72 mutations. The presence of early microglial changes in our CHMP2B mutant mice indicates neuroinflammation may be a contributing factor to the neurodegeneration observed in FTD.Frontotemporal dementia (FTD)-causing mutations in the CHMP2B gene lead to the generation of mutant C-terminally truncated CHMP2B. We report that transgenic mice expressing endogenous levels of mutant CHMP2B developed late-onset The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. The authors wish it to be known that, in their opinion, Peter L. Oliver, Diego Gomez-Nicola and Adrian M. Isaacs should be regarded as Joint Senior

Determination of Odor Detection Threshold in the Gottingen Minipig

The aim of the study was to examine the ability of Göttingen minipigs to acquire an olfaction-based operant conditioning task and to determine the detection threshold for ethyl acetate and ethanol. We used an automated olfactometer developed for rodents to train and test 14 pigs. Odor sampling and reliable responding were obtained after three to fifteen 160-trial sessions. Successful transfer of the task from ethyl acetate to ethanol was achieved in 1–4 sessions. Detection threshold for ethyl acetate varied between 10−2% and 10−6% v/v and for ethanol between 0.1% and 5 × 10−6% v/v. The results provide evidence that minipigs can successfully acquire 2-odorant discrimination using a food-rewarded instrumental conditioning paradigm for testing olfactory function. This olfactory discrimination paradigm provides reliable measures of olfactory sensitivity and thereby enables detection of changes in olfaction in a porcine model of Alzheimers disease currently being developed.

Expression of the Alzheimer’s Disease Mutations AβPP695sw and PSEN1M146I in Double-Transgenic Göttingen Minipigs

Mutations in the amyloid-β protein precursor gene (AβPP), the presenilin 1 gene (PSEN1) or the presenilin 2 gene (PSEN2) that increase production of the AβPP-derived peptide Aβ42 cause early-onset Alzheimers disease. Rodent models of the disease show that further increase in Aβ42 production and earlier brain pathology can be obtained by coexpressing AβPP and PSEN1 mutations. To generate such elevated Aβ42 level in a large animal model, we produced Göttingen minipigs carrying in their genome one copy of a human PSEN1 cDNA with the Met146Ile (PSEN1M146I) mutation and three copies of a human AβPP695 cDNA with the Lys670Asn/Met671Leu (AβPPsw) double-mutation. Both transgenes were expressed in fibroblasts and in the brain, and their respective proteins were processed normally. Immunohistochemical staining with Aβ42-specific antibodies detected intraneuronal accumulation of Aβ42 in brains from a 10- and an 18-month-old pig. Such accumulation may represent an early event in the pathogenesis of Alzheimers disease.