Alzheimer's disease associated isoforms of human CD33 distinctively modulate microglial cell responses in 5XFAD mice.

Microglia play diverse pathophysiological roles in Alzheimer's disease (AD), with genetic susceptibility factors skewing microglial cell function to influence AD risk. CD33 is an immunomodulatory receptor associated with AD susceptibility through a single nucleotide polymorphism that modulates mRNA splicing, skewing protein expression from a long protein isoform (CD33M) to a short isoform (CD33m). Understanding how human CD33 isoforms differentially impact microglial cell function in vivo has been challenging due to functional divergence of CD33 between mice and humans.

Microglial APOE4: more is less and less is more.

Apolipoprotein E (APOE) is the single greatest genetic risk factor for late onset Alzheimer's disease (AD). Yet, the cell-specific effects of APOE on microglia function have remained unclear. Fortunately, two comprehensive new studies published in the latest issue of Nature Immunology have employed complementary gain-of-function and loss-of-function approaches to provide critical new insight into the impact of microglial APOE on AD pathogenesis.

Application of N-Terminal Labeling Methods Provide Novel Insights into Endoproteolysis of the Prion Protein .

Alternative α- and β-cleavage events in the cellular prion protein (PrP) central region generate fragments with distinct biochemical features that affect prion disease pathogenesis, but the assignment of precise cleavage positions has proven challenging. Exploiting mouse transgenic models expressing wild-type (WT) PrP and an octarepeat region mutant allele (S3) with increased β-fragmentation, cleavage sites were defined using LC-MS/MS in conjunction with N-terminal enzymatic labeling and chemical in-gel acetylation. Our studies profile the net proteolytic repertoire of the adult brain, as deduced from defining hundreds of proteolytic events in other proteins, and position individual cleavage events in PrP α- and β-target areas imputed from earlier, lower resolution methods; these latter analyses established site heterogeneity, with six cleavage sites positioned in the β-cleavage region of WT PrP and nine positions for S3 PrP.

Beta-endoproteolysis of the cellular prion protein by dipeptidyl peptidase-4 and fibroblast activation protein.

The cellular prion protein (PrP) converts to alternatively folded pathogenic conformations (PrP) in prion infections and binds neurotoxic oligomers formed by amyloid-β α-synuclein, and tau. β-Endoproteolysis, which splits PrP into N- and C-terminal fragments (N2 and C2, respectively), is of interest because a protease-resistant, C2-sized fragment (C2) accumulates in the brain during prion infections, seemingly comprising the majority of PrP at disease endpoint in mice. However, candidates for the underlying proteolytic mechanism(s) remain unconfirmed in vivo.

Streamlined high-throughput cloning protocol to generate arrayed mutant libraries.

Large-scale, site-directed mutagenesis enables rapid characterization of the biochemical and biological properties of proteins. Here, we present a cost-effective and adaptable cloning pipeline to generate arrayed gene libraries for a construct of interest. We detail steps to use an open access web app to automate the design of mutagenesis primers optimized for our cloning protocols in a 96-well plate format.

CD33 isoforms in microglia and Alzheimer's disease: Friend and foe.

Alzheimer's disease (AD) is the most common form of neurodegenerative disease and is considered the main cause of dementia worldwide. Genome-wide association studies combined with integrated analysis of functional datasets support a critical role for microglia in AD pathogenesis, identifying them as important potential therapeutic targets. The ability of immunomodulatory receptors on microglia to control the response to pathogenic amyloid-β aggregates has gained significant interest.

Increasing phagocytosis of microglia by targeting CD33 with liposomes displaying glycan ligands.

CD33 is an immunomodulatory receptor expressed by microglia and genetically linked to Alzheimer's disease (AD) susceptibility. While antibodies targeting CD33 have entered clinical trials to treat neurodegeneration, it is unknown whether the glycan-binding properties of CD33 can be exploited to modulate microglia. Here, we use liposomes that multivalently display glycan ligands of CD33 (CD33L liposomes) to engage CD33.

The CD33 short isoform is a gain-of-function variant that enhances Aβ phagocytosis in microglia.

Background: CD33 is genetically linked to Alzheimer's disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an AD-protective CD33 allele (rs12459419T), is unknown. Here, we test whether hCD33m represents a loss-of-function or gain-of-function variant.

Cellular Biology of Tau Diversity and Pathogenic Conformers.

Tau accumulation is a prominent feature in a variety of neurodegenerative disorders and remarkable effort has been expended working out the biochemistry and cell biology of this cytoplasmic protein. Tau's wayward properties may derive from germline mutations in the case of frontotemporal lobar degeneration (FTLD-MAPT) but may also be prompted by less understood cues-perhaps environmental or from molecular damage as a consequence of chronological aging-in the case of idiopathic tauopathies. Tau properties are undoubtedly affected by its covalent structure and in this respect tau protein is not only subject to changes in length produced by alternative splicing and endoproteolysis, but different types of posttranslational modifications that affect different amino acid residues.

Quaternary Structure Changes for PrP Predate PrP Downregulation and Neuronal Death During Progression of Experimental Scrapie Disease.

Prion diseases are fatal neurodegenerative diseases in mammals with the unique characteristics of misfolding and aggregation of the cellular prion protein (PrP) to the scrapie prion (PrP). Although neuroinflammation and neuronal loss feature within the disease process, the details of PrP/PrP molecular transition to generate different aggregated species, and the correlation between each species and sequence of cellular events in disease pathogenesis are not fully understood. In this study, using mice inoculated with the RML isolate of mouse-adapted scrapie as a model, we applied asymmetric flow field-flow fractionation to monitor PrP and PrP particle sizes and we also measured seeding activity and resistance to proteases.

Metabolomic study of disease progression in scrapie prion infected mice; validation of a novel method for brain metabolite extraction.

Introduction: Prion disease is a form of neurodegenerative disease caused by the misfolding and aggregation of cellular prion protein (PrP). The neurotoxicity of the misfolded form of prion protein, PrP still remains understudied. Here we try to investigate this issue using a metabolomics approach.

Diverse, evolving conformer populations drive distinct phenotypes in frontotemporal lobar degeneration caused by the same MAPT-P301L mutation.

Tau protein accumulation is a common denominator of major dementias, but this process is inhomogeneous, even when triggered by the same germline mutation. We considered stochastic misfolding of human tau conformers followed by templated conversion of native monomers as an underlying mechanism and derived sensitive conformational assays to test this concept. Assessments of brains from aged TgTau transgenic mice revealed a prodromal state and three distinct signatures for misfolded tau.

The CNS in inbred transgenic models of 4-repeat Tauopathy develops consistent tau seeding capacity yet focal and diverse patterns of protein deposition.

Background: MAPT mutations cause neurodegenerative diseases such as frontotemporal dementia but, strikingly, patients with the same mutation may have different clinical phenotypes.

Methods: Given heterogeneities observed in a transgenic (Tg) mouse line expressing low levels of human (2 N, 4R) P301L Tau, we backcrossed founder stocks of mice to C57BL/6Tac, 129/SvEvTac and FVB/NJ inbred backgrounds to discern the role of genetic versus environmental effects on disease-related phenotypes.

Results: Three inbred derivatives of a TgTau founder line had similar quality and steady-state quantity of Tau production, accumulation of abnormally phosphorylated 64-68 kDa Tau species from 90 days of age onwards and neuronal loss in aged Tg mice.

Microtubule Dynamicity Is More Important than Stability in Memory Formation: an In Vivo Study.

It has been shown that microtubule (MT) activity and dynamics can have huge impacts on synaptic plasticity and memory formation. This is mainly due to various functions of MTs in neurons; MTs are involved in dendritic spine formation, axonal transportation, neuronal polarity, and receptor trafficking. Recent studies from our group and other labs have suggested the possible role of brain MT dynamicity and activity in memory; however, there is a need for more detailed studies regarding this aspect.

Chronic, long-term social stress can cause decreased microtubule protein network activity and dynamics in cerebral cortex of male Wistar rats.

Social stress is viewed as a factor in the etiology of a variety of psychopathologies such as depression and anxiety. Animal models of social stress are well developed and widely used in studying clinical and physiological effects of stress. Stress is known to significantly affect learning and memory, and this effect strongly depends on the type of stress, its intensity, and duration.