Higher Levels of Stress and Neuropsychological Symptoms Are Associated With a High Nausea Profile in Patients With Cancer Receiving Chemotherapy.

OBJECTIVES: To evaluate differences in the severity of global, cancer-specific, and cumulative life stress, resilience, and common neuropsychological symptoms among four subgroups of patients with distinct chemotherapy-induced nausea (CIN) profiles. SAMPLE & SETTING: Adult patients with cancer (N = 1,343) receiving chemotherapy. METHODS & VARIABLES: Patients completed stress, resilience, and neuropsychological symptom severity measures. The Memorial Symptom Assessment Scale was used to assess CIN occurrence six times over two cycles of chemotherapy. Parametric and nonparametric statistics were used to evaluate differences among subgroups of patients with distinct CIN profiles. RESULTS: The high class had significantly higher levels of global, cancer-specific, and cumulative life stress; significantly higher levels of depression, anxiety, sleep disturbance, morning and evening fatigue, and pain; and lower levels of morning and evening energy and cognitive dysfunction. IMPLICATIONS FOR NURSING: Clinicians need to evaluate CIN occurrence across each cycle of chemotherapy and assess patients for various types of stress and common neuropsychological symptoms.

Plasma PolyQ-ATXN3 Levels Associate With Cerebellar Degeneration and Behavioral Abnormalities in a New AAV-Based SCA3 Mouse Model.

Spinocerebellar ataxia type 3 (SCA3) is a dominantly inherited cerebellar ataxia caused by the expansion of a polyglutamine (polyQ) repeat in the gene encoding ATXN3. The polyQ expansion induces protein inclusion formation in the neurons of patients and results in neuronal degeneration in the cerebellum and other brain regions. We used adeno-associated virus (AAV) technology to develop a new mouse model of SCA3 that recapitulates several features of the human disease, including locomotor defects, cerebellar-specific neuronal loss, polyQ-expanded ATXN3 inclusions, and TDP-43 pathology. We also found that neurofilament light is elevated in the cerebrospinal fluid (CSF) of the SCA3 animals, and the expanded polyQ-ATXN3 protein can be detected in the plasma. Interestingly, the levels of polyQ-ATXN3 in plasma correlated with measures of cerebellar degeneration and locomotor deficits in 6-month-old SCA3 mice, supporting the hypothesis that this factor could act as a biomarker for SCA3.

Culture shock: microglial heterogeneity, activation, and disrupted single-cell microglial networks in vitro.

BACKGROUND: Microglia, the resident immune cells of the brain, play a critical role in numerous diseases, but are a minority cell type and difficult to genetically manipulate in vivo with viral vectors and other approaches. Primary cultures allow a more controlled setting to investigate these cells, but morphological and transcriptional changes upon removal from their normal brain environment raise many caveats from in vitro studies. METHODS: To investigate whether cultured microglia recapitulate in vivo microglial signatures, we used single-cell RNA sequencing (scRNAseq) to compare microglia freshly isolated from the brain to primary microglial cultures. We performed cell population discovery, differential expression analysis, and gene co-expression module analysis to compare signatures between in vitro and in vivo microglia. We constructed causal predictive network models of transcriptional regulators from the scRNAseq data and identified a set of potential key drivers of the cultured phenotype. To validate this network analysis, we knocked down two of these key drivers, C1qc and Prdx1, in primary cultured microglia and quantified changes in microglial activation markers. RESULTS: We found that, although often assumed to be a relatively homogenous population of cells in culture, in vitro microglia are a highly heterogeneous population consisting of distinct subpopulations of cells with transcriptional profiles reminiscent of macrophages and monocytes, and are marked by transcriptional programs active in neurodegeneration and other disease states. We found that microglia in vitro presented transcriptional activation of a set of "culture shock genes" not found in freshly isolated microglia, characterized by strong upregulation of disease-associated genes including Apoe, Lyz2, and Spp1, and downregulation of homeostatic microglial markers, including Cx3cr1, P2ry12, and Tmem119. Finally, we found that cultured microglia prominently alter their transcriptional machinery modulated by key drivers from the homeostatic to activated phenotype. Knockdown of one of these drivers, C1qc, resulted in downregulation of microglial activation genes Lpl, Lyz2, and Ccl4. CONCLUSIONS: Overall, our data suggest that when removed from their in vivo home environment, microglia suffer a severe case of "culture shock", drastically modulating their transcriptional regulatory network state from homeostatic to activated through upregulation of modules of culture-specific genes. Consequently, cultured microglia behave as a disparate cell type that does not recapitulate the homeostatic signatures of microglia in vivo. Finally, our predictive network model discovered potential key drivers that may convert activated microglia back to their homeostatic state, allowing for more accurate representation of in vivo states in culture. Knockdown of key driver C1qc partially attenuated microglial activation in vitro, despite C1qc being only weakly upregulated in culture. This suggests that even genes that are not strongly differentially expressed across treatments or preparations may drive downstream transcriptional changes in culture.

Widespread choroid plexus contamination in sampling and profiling of brain tissue.

The choroid plexus, a tissue responsible for producing cerebrospinal fluid, is found predominantly in the lateral and fourth ventricles of the brain. This highly vascularized and ciliated tissue is made up of specialized epithelial cells and capillary networks surrounded by connective tissue. Given the complex structure of the choroid plexus, this can potentially result in contamination during routine tissue dissection. Bulk and single-cell RNA sequencing studies, as well as genome-wide in situ hybridization experiments (Allen Brain Atlas), have identified several canonical markers of choroid plexus such as Ttr, Folr1, and Prlr. We used the Ttr gene as a marker to query the Gene Expression Omnibus database for transcriptome studies of brain tissue and identified at least some level of likely choroid contamination in numerous studies that could have potentially confounded data analysis and interpretation. We also analyzed transcriptomic datasets from human samples from Allen Brain Atlas and the Genotype-Tissue Expression (GTEx) database and found abundant choroid contamination, with regions in closer proximity to choroid more likely to be impacted such as hippocampus, cervical spinal cord, substantia nigra, hypothalamus, and amygdala. In addition, analysis of both the Allen Brain Atlas and GTEx datasets for differentially expressed genes between likely "high contamination" and "low contamination" groups revealed a clear enrichment of choroid plexus marker genes and gene ontology pathways characteristic of these ciliated choroid cells. Inclusion of these contaminated samples could result in biological misinterpretation or simply add to the statistical noise and mask true effects. We cannot assert that Ttr or other genes/proteins queried in targeted assays are artifacts from choroid contamination as some of these differentials may be due to true biological effects. However, for studies that have an unequal distribution of choroid contamination among groups, investigators may wish to remove contaminated samples from analyses or incorporate choroid marker gene expression into their statistical modeling. In addition, we suggest that a simple RT-qPCR or western blot for choroid markers would mitigate unintended choroid contamination for any experiment, but particularly for samples intended for more costly omic profiling. This study highlights an unexpected problem for neuroscientists, but it is also quite possible that unintended contamination of adjacent structures occurs during dissections for other tissues but has not been widely recognized.

Urine levels of the polyglutamine ataxin-3 protein are elevated in patients with spinocerebellar ataxia type 3.

INTRODUCTION: Accumulation of polyglutamine (polyQ) ataxin-3 (ATXN3) contributes to the pathobiology of spinocerebellar ataxia type 3 (SCA3). Recently, we showed that polyQ ATXN3 is elevated in the plasma and cerebrospinal fluid (CSF) of SCA3 patients, and has the potential to serve as a biological marker for this disease [1]. Based on these findings, we investigated whether polyQ ATXN3 can also be detected in urine samples from SCA3 patients. METHODS: We analyzed urine samples from 30 SCA3 subjects (including one pre-symptomatic subject), 35 subjects with other forms of ataxia, and 37 healthy controls. To quantify polyQ ATXN3 protein levels, we used our previously developed immunoassay. RESULTS: PolyQ ATXN3 can be detected in the urine of SCA3 patients, but not in urine samples from healthy controls or other forms of ataxia. There was a significant statistical association between polyQ ATXN3 levels in urine samples and those in plasma. Further, the levels of polyQ ATXN3 urine associated with an earlier age of SCA3 disease onset. CONCLUSION: As clinical trials for SCA3 advance, urine polyQ ATXN3 protein has potential to be a useful, non-invasive and inexpensive biomarker for SCA3.

Estrous Cycle Modulation of Feeding and Relaxin-3/Rxfp3 mRNA Expression: Implications for Estradiol Action.

INTRODUCTION: Food intake varies during the ovarian hormone/estrous cycle in humans and rodents, an effect mediated mainly by estradiol. A potential mediator of the central anorectic effects of estradiol is the neuropeptide relaxin-3 (RLN3) synthetized in the nucleus incertus (NI) and acting via the relaxin family peptide-3 receptor (RXFP3). METHODS: We investigated the relationship between RLN3/RXFP3 signaling and feeding behavior across the female rat estrous cycle. We used in situ hybridization to investigate expression patterns of Rln3 mRNA in NI and Rxfp3 mRNA in the hypothalamic paraventricular nucleus (PVN), lateral hypothalamic area (LHA), medial preoptic area (MPA), and bed nucleus of the stria terminalis (BNST), across the estrous cycle. We identified expression of estrogen receptors (ERs) in the NI using droplet digital PCR and assessed the electrophysiological responsiveness of NI neurons to estradiol in brain slices. RESULTS: Rln3 mRNA reached the lowest levels in the NI pars compacta during proestrus. Rxfp3 mRNA levels varied across the estrous cycle in a region-specific manner, with changes observed in the perifornical LHA, magnocellular PVN, dorsal BNST, and MPA, but not in the parvocellular PVN or lateral LHA. G protein-coupled estrogen receptor 1 (Gper1) mRNA was the most abundant ER transcript in the NI. Estradiol inhibited 33% of type 1 NI neurons, including RLN3-positive cells. CONCLUSION: These findings demonstrate that the RLN3/RXFP3 system is modulated by the estrous cycle, and although further studies are required to better elucidate the cellular and molecular mechanisms of estradiol signaling, current results implicate the involvement of the RLN3/RXFP3 system in food intake fluctuations observed across the estrous cycle in female rats.

Astrocyte-derived clusterin suppresses amyloid formation in vivo.

BACKGROUND: Accumulation of amyloid-ß (Aß) peptide in the brain is a pathological hallmark of Alzheimer's disease (AD). The clusterin (CLU) gene confers a risk for AD and CLU is highly upregulated in AD patients, with the common non-coding, protective CLU variants associated with increased expression. Although there is strong evidence implicating CLU in amyloid metabolism, the exact mechanism underlying the CLU involvement in AD is not fully understood or whether physiologic alterations of CLU levels in the brain would be protective. RESULTS: We used a gene delivery approach to overexpress CLU in astrocytes, the major source of CLU expression in the brain. We found that CLU overexpression resulted in a significant reduction of total and fibrillar amyloid in both cortex and hippocampus in the APP/PS1 mouse model of AD amyloidosis. CLU overexpression also ameliorated amyloid-associated neurotoxicity and gliosis. To complement these overexpression studies, we also analyzed the effects of haploinsufficiency of Clu using heterozygous (Clu+/-) mice and control littermates in the APP/PS1 model. CLU reduction led to a substantial increase in the amyloid plaque load in both cortex and hippocampus in APP/PS1; Clu+/- mice compared to wild-type (APP/PS1; Clu+/+) littermate controls, with a concomitant increase in neuritic dystrophy and gliosis. CONCLUSIONS: Thus, both physiologic ~ 30% overexpression or ~ 50% reduction in CLU have substantial impacts on amyloid load and associated pathologies. Our results demonstrate that CLU plays a major role in Aß accumulation in the brain and suggest that efforts aimed at CLU upregulation via pharmacological or gene delivery approaches offer a promising therapeutic strategy to regulate amyloid pathology.

APOE4 exacerbates α-synuclein pathology and related toxicity independent of amyloid.

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease mainly by driving amyloid-ß pathology. Recently, APOE4 has also been found to be a genetic risk factor for Lewy body dementia (LBD), which includes dementia with Lewy bodies and Parkinson's disease dementia. How APOE4 drives risk of LBD and whether it has a direct effect on α-synuclein pathology are not clear. Here, we generated a mouse model of synucleinopathy using an adeno-associated virus gene delivery of α-synuclein in human APOE-targeted replacement mice expressing APOE2, APOE3, or APOE4. We found that APOE4, but not APOE2 or APOE3, increased α-synuclein pathology, impaired behavioral performances, worsened neuronal and synaptic loss, and increased astrogliosis at 9 months of age. Transcriptomic profiling in APOE4-expressing α-synuclein mice highlighted altered lipid and energy metabolism and synapse-related pathways. We also observed an effect of APOE4 on α-synuclein pathology in human postmortem brains with LBD and minimal amyloid pathology. Our data demonstrate a pathogenic role of APOE4 in exacerbating α-synuclein pathology independent of amyloid, providing mechanistic insights into how APOE4 increases the risk of LBD.

Aberrant deposition of stress granule-resident proteins linked to C9orf72-associated TDP-43 proteinopathy.

BACKGROUND: A G4C2 hexanucleotide repeat expansion in the noncoding region of C9orf72 is the major genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis (c9FTD/ALS). Putative disease mechanisms underlying c9FTD/ALS include toxicity from sense G4C2 and antisense G2C4 repeat-containing RNA, and from dipeptide repeat (DPR) proteins unconventionally translated from these RNA products. METHODS: Intracerebroventricular injections with adeno-associated virus (AAV) encoding 2 or 149 G4C2 repeats were performed on postnatal day 0, followed by assessment of behavioral and neuropathological phenotypes. RESULTS: Relative to control mice, gliosis and neurodegeneration accompanied by cognitive and motor deficits were observed in (G4C2)149 mice by 6 months of age. Recapitulating key pathological hallmarks, we also demonstrate that sense and antisense RNA foci, inclusions of poly(GA), poly(GP), poly(GR), poly(PR), and poly(PA) DPR proteins, and inclusions of endogenous phosphorylated TDP-43 (pTDP-43) developed in (G4C2)149 mice but not control (G4C2)2 mice. Notably, proteins that play a role in the regulation of stress granules - RNA-protein assemblies that form in response to translational inhibition and that have been implicated in c9FTD/ALS pathogenesis - were mislocalized in (G4C2)149 mice as early as 3 months of age. Specifically, we observed the abnormal deposition of stress granule components within inclusions immunopositive for poly(GR) and pTDP-43, as well as evidence of nucleocytoplasmic transport defects. CONCLUSIONS: Our in vivo model of c9FTD/ALS is the first to robustly recapitulate hallmark features derived from both sense and antisense C9orf72 repeat-associated transcripts complete with neurodegeneration and behavioral impairments. More importantly, the early appearance of persistent pathological stress granules prior to significant pTDP-43 deposition implicates an aberrant stress granule response as a key disease mechanism driving TDP-43 proteinopathy in c9FTD/ALS.

Enhanced phosphorylation of T153 in soluble tau is a defining biochemical feature of the A152T tau risk variant.

Pathogenic mutations in the tau gene (microtubule associated protein tau, MAPT) are linked to the onset of tauopathy, but the A152T variant is unique in acting as a risk factor for a range of disorders including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and dementia with Lewy bodies (DLB). In order to provide insight into the mechanism by which A152T modulates disease risk, we developed a novel mouse model utilizing somatic brain transgenesis with adeno-associated virus (AAV) to drive tau expression in vivo, and validated the model by confirming the distinct biochemical features of A152T tau in postmortem brain tissue from human carriers. Specifically, TauA152T-AAV mice exhibited increased tau phosphorylation that unlike animals expressing the pathogenic P301L mutation remained localized to the soluble fraction. To investigate the possibility that the A152T variant might alter the phosphorylation state of tau on T152 or the neighboring T153 residue, we generated a novel antibody that revealed significant accumulation of soluble tau species that were hyperphosphorylated on T153 (pT153) in TauA152T-AAV mice, which were absent the soluble fraction of TauP301L-AAV mice. Providing new insight into the role of A152T in modifying risk of tauopathy, as well as validating the TauA152T-AAV model, we demonstrate that the presence of soluble pT153-positive tau species in human postmortem brain tissue differentiates A152T carriers from noncarriers, independent of disease classification. These results implicate both phosphorylation of T153 and an altered solubility profile in the mechanism by which A152T modulates disease risk.

APOE ε2 is associated with increased tau pathology in primary tauopathy.

Apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease mainly by modulating amyloid-ß pathology. APOE ε4 is also shown to exacerbate neurodegeneration and neuroinflammation in a tau transgenic mouse model. To further evaluate the association of APOE genotype with the presence and severity of tau pathology, we express human tau via an adeno-associated virus gene delivery approach in human APOE targeted replacement mice. We find increased hyperphosphorylated tau species, tau aggregates, and behavioral abnormalities in mice expressing APOE ε2/ε2. We also show that in humans, the APOE ε2 allele is associated with increased tau pathology in the brains of progressive supranuclear palsy (PSP) cases. Finally, we identify an association between the APOE ε2/ε2 genotype and risk of tauopathies using two series of pathologically-confirmed cases of PSP and corticobasal degeneration. Our data together suggest APOE ε2 status may influence the risk and progression of tauopathy.

Microglial translational profiling reveals a convergent APOE pathway from aging, amyloid, and tau.

Alzheimer's disease (AD) is an age-associated neurodegenerative disease characterized by amyloidosis, tauopathy, and activation of microglia, the brain resident innate immune cells. We show that a RiboTag translational profiling approach can bypass biases due to cellular enrichment/cell sorting. Using this approach in models of amyloidosis, tauopathy, and aging, we revealed a common set of alterations and identified a central APOE-driven network that converged on CCL3 and CCL4 across all conditions. Notably, aged females demonstrated a significant exacerbation of many of these shared transcripts in this APOE network, revealing a potential mechanism for increased AD susceptibility in females. This study has broad implications for microglial transcriptomic approaches and provides new insights into microglial pathways associated with different pathological aspects of aging and AD.

Loss of Tmem106b is unable to ameliorate frontotemporal dementia-like phenotypes in an AAV mouse model of C9ORF72-repeat induced toxicity.

Loss-of-function mutations in progranulin (GRN) and a non-coding (GGGGCC)n hexanucleotide repeat expansions in C9ORF72 are the two most common genetic causes of frontotemporal lobar degeneration with aggregates of TAR DNA binding protein 43 (FTLD-TDP). TMEM106B encodes a type II transmembrane protein with unknown function. Genetic variants in TMEM106B associated with reduced TMEM106B levels have been identified as disease modifiers in individuals with GRN mutations and C9ORF72 expansions. Recently, loss of Tmem106b has been reported to protect the FTLD-like phenotypes in Grn-/- mice. Here, we generated Tmem106b-/- mice and examined whether loss of Tmem106b could rescue FTLD-like phenotypes in an AAV mouse model of C9ORF72-repeat induced toxicity. Our results showed that neither partial nor complete loss of Tmem106b was able to rescue behavioral deficits induced by the expression of (GGGGCC)66 repeats (66R). Loss of Tmem106b also failed to ameliorate 66R-induced RNA foci, dipeptide repeat protein formation and pTDP-43 pathological burden. We further found that complete loss of Tmem106b increased astrogliosis, even in the absence of 66R, and failed to rescue 66R-induced neuronal cell loss, whereas partial loss of Tmem106b significantly rescued the neuronal cell loss but not neuroinflammation induced by 66R. Finally, we showed that overexpression of 66R did not alter expression of Tmem106b and other lysosomal genes in vivo, and subsequent analyses in vitro found that transiently knocking down C9ORF72, but not overexpression of 66R, significantly increased TMEM106B and other lysosomal proteins. In summary, reducing Tmem106b levels failed to rescue FTLD-like phenotypes in a mouse model mimicking the toxic gain-of-functions associated with overexpression of 66R. Combined with the observation that loss of C9ORF72 and not 66R overexpression was associated with increased levels of TMEM106B, this work suggests that the protective TMEM106B haplotype may exert its effect in expansion carriers by counteracting lysosomal dysfunction resulting from a loss of C9ORF72.

Potentially Modifiable Risk Factors for Long-Term Cognitive Impairment After Critical Illness: A Systematic Review.

Long-term cognitive impairment is common in survivors of critical illness. Little is known about the etiology of this serious complication. We sought to summarize current scientific knowledge about potentially modifiable risk factors during intensive care unit (ICU) treatment that may play a substantial role in the development of long-term cognitive impairment. All searches were run on October 1, 2017. The search strategy included Ovid MEDLINE, Ovid Embase, Ovid CDR, Cochrane Central Register of Controlled Trials and Database of Abstracts of Reviews of Effect, Scopus, and Web of Science, and included MeSH headings and keywords related to intensive care, critical care, and cognitive disorders. Searches were restricted to adult subjects. Inclusion required follow-up cognitive evaluation at least 2 months after ICU discharge. Studies assessing patients with cardiac arrest, traumatic brain injury, and cardiac surgery history were excluded. The search strategy resulted in 3180 studies. Of these, 28 studies (.88%) met our inclusion criteria and were analyzed. Delirium and duration of delirium were associated with long-term cognitive impairment after ICU admission in 6 of 9 studies in which this factor was analyzed. Weaker and more inconsistent associations have been reported with hypoglycemia, hyperglycemia, fluctuations in serum glucose levels, and in-hospital acute stress symptoms. Instead, most of the studies did not find significant associations between long-term cognitive impairment and mechanical ventilation; use of sedatives, vasopressors, or analgesic medications; enteral feeding; hypoxia; extracorporeal membrane oxygenation; systolic blood pressure; pulse rate; or length of ICU stay. Prolonged delirium may be a risk factor for long-term cognitive impairment after critical illness, though this association has not been entirely consistent across studies. Other potentially preventable factors have not been shown to have strong or consistent associations with long-term cognitive dysfunction in survivors of critical illness.

TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are age-related neurodegenerative disorders with shared genetic etiologies and overlapping clinical and pathological features. Here we studied a novel ALS/FTD family and identified the P362L mutation in the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1). Subsequent genetic association analyses showed an increased burden of TIA1 LCD mutations in ALS patients compared to controls (p = 8.7 × 10-6). Postmortem neuropathology of five TIA1 mutations carriers showed a consistent pathological signature with numerous round, hyaline, TAR DNA-binding protein 43 (TDP-43)-positive inclusions. TIA1 mutations significantly increased the propensity of TIA1 protein to undergo phase transition. In live cells, TIA1 mutations delayed stress granule (SG) disassembly and promoted the accumulation of non-dynamic SGs that harbored TDP-43. Moreover, TDP-43 in SGs became less mobile and insoluble. The identification of TIA1 mutations in ALS/FTD reinforces the importance of RNA metabolism and SG dynamics in ALS/FTD pathogenesis.

Loss of clusterin shifts amyloid deposition to the cerebrovasculature via disruption of perivascular drainage pathways.

Alzheimer's disease (AD) is characterized by amyloid-ß (Aß) peptide deposition in brain parenchyma as plaques and in cerebral blood vessels as cerebral amyloid angiopathy (CAA). CAA deposition leads to several clinical complications, including intracerebral hemorrhage. The underlying molecular mechanisms that regulate plaque and CAA deposition in the vast majority of sporadic AD patients remain unclear. The clusterin (CLU) gene is genetically associated with AD and CLU has been shown to alter aggregation, toxicity, and blood-brain barrier transport of Aß, suggesting it might play a key role in regulating the balance between Aß deposition and clearance in both brain and blood vessels. Here, we investigated the effect of CLU on Aß pathology using the amyloid precursor protein/presenilin 1 (APP/PS1) mouse model of AD amyloidosis on a Clu+/+ or Clu-/- background. We found a marked decrease in plaque deposition in the brain parenchyma but an equally striking increase in CAA within the cerebrovasculature of APP/PS1;Clu-/- mice. Surprisingly, despite the several-fold increase in CAA levels, APP/PS1;Clu-/- mice had significantly less hemorrhage and inflammation. Mice lacking CLU had impaired clearance of Aß in vivo and exogenously added CLU significantly prevented Aß binding to isolated vessels ex vivo. These findings suggest that in the absence of CLU, Aß clearance shifts to perivascular drainage pathways, resulting in fewer parenchymal plaques but more CAA because of loss of CLU chaperone activity, complicating the potential therapeutic targeting of CLU for AD.

Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain.

Abnormal accumulation of alpha-synuclein (αsyn) is a pathological hallmark of Lewy body related disorders such as Parkinson's disease and Dementia with Lewy body disease. During the past two decades, a myriad of animal models have been developed to mimic pathological features of synucleinopathies by over-expressing human αsyn. Although different strategies have been used, most models have little or no reliable and predictive phenotype. Novel animal models are a valuable tool for understanding neuronal pathology and to facilitate development of new therapeutics for these diseases. Here, we report the development and characterization of a novel model in which mice rapidly express wild-type αsyn via somatic brain transgenesis mediated by adeno-associated virus (AAV). At 1, 3, and 6 months of age following intracerebroventricular (ICV) injection, mice were subjected to a battery of behavioral tests followed by pathological analyses of the brains. Remarkably, significant levels of αsyn expression are detected throughout the brain as early as 1 month old, including olfactory bulb, hippocampus, thalamic regions and midbrain. Immunostaining with a phospho-αsyn (pS129) specific antibody reveals abundant pS129 expression in specific regions. Also, pathologic αsyn is detected using the disease specific antibody 5G4. However, this model did not recapitulate behavioral phenotypes characteristic of rodent models of synucleinopathies. In fact no deficits in motor function or cognition were observed at 3 or 6 months of age. Taken together, these findings show that transduction of neonatal mouse with AAV-αsyn can successfully lead to rapid, whole brain transduction of wild-type human αsyn, but increased levels of wildtype αsyn do not induce behavior changes at an early time point (6 months), despite pathological changes in several neurons populations as early as 1 month.

Soluble TREM2 induces inflammatory responses and enhances microglial survival.

Triggering receptor expressed on myeloid cells 2 (TREM2) is an innate immune receptor expressed in microglia in the brain. A soluble form of TREM2 (sTREM2) derived from proteolytic cleavage of the cell surface receptor is increased in the preclinical stages of AD and positively correlates with the amounts of total and phosphorylated tau in the cerebrospinal fluid. However, the physiological and pathological functions of sTREM2 remain unknown. Here, we show that sTREM2 promotes microglial survival in a PI3K/Akt-dependent manner and stimulates the production of inflammatory cytokines depending on NF-κB. Variants of sTREM2 carrying AD risk-associated mutations were less potent in both suppressing apoptosis and triggering inflammatory responses. Importantly, sTREM2 delivered to the hippocampi of both wild-type and Trem2-knockout mice elevated the expression of inflammatory cytokines and induced morphological changes of microglia. Collectively, these data indicate that sTREM2 triggers microglial activation inducing inflammatory responses and promoting survival. This study has implications for the pathogenesis of AD and provides insights into targeting sTREM2 pathway for AD therapy.

TREM2 Promotes Microglial Survival by Activating Wnt/ß-Catenin Pathway.

Triggering Receptor Expressed on Myeloid cells 2 (TREM2), which is expressed on myeloid cells including microglia in the CNS, has recently been identified as a risk factor for Alzheimer's disease (AD). TREM2 transmits intracellular signals through its transmembrane binding partner DNAX-activating protein 12 (DAP12). Homozygous mutations inactivating TREM2 or DAP12 lead to Nasu-Hakola disease; however, how AD risk-conferring variants increase AD risk is not clear. To elucidate the signaling pathways underlying reduced TREM2 expression or loss of function in microglia, we respectively knocked down and knocked out the expression of TREM2 in in vitro and in vivo models. We found that TREM2 deficiency reduced the viability and proliferation of primary microglia, reduced microgliosis in Trem2-/- mouse brains, induced cell cycle arrest at the G1/S checkpoint, and decreased the stability of ß-catenin, a key component of the canonical Wnt signaling pathway responsible for maintaining many biological processes, including cell survival. TREM2 stabilized ß-catenin by inhibiting its degradation via the Akt/GSK3ß signaling pathway. More importantly, treatment with Wnt3a, LiCl, or TDZD-8, which activates the ß-catenin-mediated Wnt signaling pathway, rescued microglia survival and microgliosis in Trem2-/- microglia and/or in Trem2-/- mouse brain. Together, our studies demonstrate a critical role of TREM2-mediated Wnt/ß-catenin pathway in microglial viability and suggest that modulating this pathway therapeutically may help to combat the impaired microglial survival and microgliosis associated with AD.SIGNIFICANCE STATEMENT Mutations in the TREM2 (Triggering Receptor Expressed on Myeloid cells 2) gene are associated with increased risk for Alzheimer's disease (AD) with effective sizes comparable to that of the apolipoprotein E (APOE) ε4 allele, making it imperative to understand the molecular pathway(s) underlying TREM2 function in microglia. Our findings shed new light on the relationship between TREM2/DNAX-activating protein 12 (DAP12) signaling and Wnt/ß-catenin signaling and provide clues as to how reduced TREM2 function might impair microglial survival in AD pathogenesis. We demonstrate that TREM2 promotes microglial survival by activating the Wnt/ß-catenin signaling pathway and that it is possible to restore Wnt/ß-catenin signaling when TREM2 activity is disrupted or reduced. Therefore, we demonstrate the potential for manipulating the TREM2/ß-catenin signaling pathway for the treatment of AD.

Tau deposition drives neuropathological, inflammatory and behavioral abnormalities independently of neuronal loss in a novel mouse model.

Aberrant tau protein accumulation drives neurofibrillary tangle (NFT) formation in several neurodegenerative diseases. Currently, efforts to elucidate pathogenic mechanisms and assess the efficacy of therapeutic targets are limited by constraints of existing models of tauopathy. In order to generate a more versatile mouse model of tauopathy, somatic brain transgenesis was utilized to deliver adeno-associated virus serotype 1 (AAV1) encoding human mutant P301L-tau compared with GFP control. At 6 months of age, we observed widespread human tau expression with concomitant accumulation of hyperphosphorylated and abnormally folded proteinase K resistant tau. However, no overt neuronal loss was observed, though significant abnormalities were noted in the postsynaptic scaffolding protein PSD95. Neurofibrillary pathology was also detected with Gallyas silver stain and Thioflavin-S, and electron microscopy revealed the deposition of closely packed filaments. In addition to classic markers of tauopathy, significant neuroinflammation and extensive gliosis were detected in AAV1-Tau(P301L) mice. This model also recapitulates the behavioral phenotype characteristic of mouse models of tauopathy, including abnormalities in exploration, anxiety, and learning and memory. These findings indicate that biochemical and neuropathological hallmarks of tauopathies are accurately conserved and are independent of cell death in this novel AAV-based model of tauopathy, which offers exceptional versatility and speed in comparison with existing transgenic models. Therefore, we anticipate this approach will facilitate the identification and validation of genetic modifiers of disease, as well as accelerate preclinical assessment of potential therapeutic targets.