Nat Neurosci. 2019 Jan;22(1):47-56.
Fu H, Possenti A, Freer R, Nakano Y, Villegas NCH, Tang M, Cauhy PVM, Lassus BA, Chen S, Fowler SL, Figueroa HY, Huey ED, Johnson GVW, Vendruscolo M, Duff KE.
PMID: 30559469 | DOI: 10.1038/s41593-018-0298-7
Excitatory neurons are preferentially impaired in early Alzheimer's disease but the pathways contributing to their relative vulnerability remain largely unknown. Here we report that pathological tau accumulation takes place predominantly in excitatory neurons compared to inhibitory neurons, not only in the entorhinal cortex, a brain region affected in early Alzheimer's disease, but also in areas affected later by the disease. By analyzing RNA transcripts from single-nucleus RNA datasets, we identified a specific tau homeostasis signature of genes differentially expressed in excitatory compared to inhibitory neurons. One of the genes, BCL2-associated athanogene 3 (BAG3), a facilitator of autophagy, was identified as a hub, or master regulator, gene. We verified that reducing BAG3 levels in primary neurons exacerbated pathological tau accumulation, whereas BAG3 overexpression attenuated it. These results define a tau homeostasis signature that underlies the cellular and regional vulnerability of excitatory neurons to tau pathology.
Proceedings of the National Academy of Sciences of the United States of America
Dias, CM;Issac, B;Sun, L;Lukowicz, A;Talukdar, M;Akula, SK;Miller, MB;Walsh, K;Rockowitz, S;Walsh, CA;
PMID: 37252957 | DOI: 10.1073/pnas.2300052120
Short trinucleotide expansions at the FMR1 locus are associated with the late-onset condition fragile X-associated tremor/ataxia syndrome (FXTAS), which shows very different clinical and pathological features from fragile X syndrome (associated with longer expansions), with no clear molecular explanation for these marked differences. One prevailing theory posits that the shorter, premutation expansion uniquely causes extreme neurotoxic increases in FMR1 mRNA (i.e., four to eightfold increases), but evidence to support this hypothesis is largely derived from analysis of peripheral blood. We applied single-nucleus RNA sequencing to postmortem frontal cortex and cerebellum from 7 individuals with premutation and matched controls (n = 6) to assess cell type-specific molecular neuropathology. We found only modest upregulation (~1.3-fold) of FMR1 in some glial populations associated with premutation expansions. In premutation cases, we also identified decreased astrocyte proportions in the cortex. Differential expression and gene ontology analysis demonstrated altered neuroregulatory roles of glia. Using network analyses, we identified cell type-specific and region-specific patterns of FMR1 protein target gene dysregulation unique to premutation cases, with notable network dysregulation in the cortical oligodendrocyte lineage. We used pseudotime trajectory analysis to determine how oligodendrocyte development was altered and identified differences in early gene expression in oligodendrocyte trajectories in premutation cases specifically, implicating early cortical glial developmental perturbations. These findings challenge dogma regarding extremely elevated FMR1 increases in FXTAS and implicate glial dysregulation as a critical facet of premutation pathophysiology, representing potential unique therapeutic targets directly derived from the human condition.
Acta pharmacologica Sinica
Zhang, YM;Ye, LY;Li, TY;Guo, F;Guo, F;Li, Y;Li, YF;
PMID: 34811511 | DOI: 10.1038/s41401-021-00807-0
Hypidone hydrochloride (YL-0919) is a novel antidepressant in clinical phase II trial. Previous studies show that YL-0919 is a selective 5-HT (serotonin) reuptake inhibitor, 5-HT1A receptor partial agonist, and 5-HT6 receptor agonist, which exerts antidepressant effects in various animal models, but its effects on neural function remain unclear. Medial prefrontal cortex (mPFC), a highly evolved brain region, controls highest order cognitive functions and emotion regulation. In this study we investigated the effects of YL-0919 on the mPFC function, including the changes in neuronal activities using electrophysiological recordings. Extracellular recording (in vivo) showed that chronic administration of YL-0919 significantly increased the spontaneous discharges of mPFC neurons. In mouse mPFC slices, whole-cell recording revealed that perfusion of YL-0919 significantly increased the frequency of sEPSCs, but decreased the frequency of sIPSCs. Then we conducted whole-cell recording in mPFC slices of GAD67-GFP transgenic mice, and demonstrated that YL-0919 significantly inhibited the excitability of GABAergic neurons. In contrast, it did not alter the excitability of pyramidal neurons in mPFC slices of normal mice. Moreover, the inhibition of GABAergic neurons by YL-0919 was prevented by pre-treatment with 5-HT1A receptor antagonist WAY 100635. Finally, chronic administration of YL-0919 significantly increased the phosphorylation levels of mTOR and GSK-3β in the mPFC as compared with vehicle. Taken together, our results demonstrate that YL-0919 enhances the excitability of mPFC via a disinhibition mechanism to fulfill its rapid antidepressant neural mechanism, which was accomplished by 5-HT1A receptor-mediated inhibition of inhibitory GABAergic interneurons.
Biological Psychiatry Global Open Science
Jiang, S;Zhang, H;Eiden, L;
| DOI: 10.1016/j.bpsgos.2023.04.001
Background The neuropeptide PACAP is a master regulator of central and peripheral stress responses, yet it is not clear how PACAP projections throughout the brain execute endocrine and behavioral stress responses. Methods We used AAV neuronal tracing, an acute restraint stress (ARS) paradigm, and intersectional genetics, in C57Bl6 mice, to identify PACAP-containing circuits controlling stress-induced behavior and endocrine activation. Results PACAP deletion from forebrain excitatory neurons, including a projection directly from medial prefrontal cortex (mPFC) to hypothalamus, impairs c-fos activation and CRH mRNA elevation in PVN after 2 hr of restraint, without affecting ARS-induced hypophagia, or c-fos elevation in non-hypothalamic brain. Elimination of PACAP within projections from lateral parabrachial nucleus to extended amygdala (EA), on the other hand, attenuates ARS-induced hypophagia, along with EA fos induction, without affecting ARS-induced CRH mRNA elevation in PVN. PACAP projections to EA terminate at PKCδ neurons in both central amygdala (CeA) and oval nuclei of bed nucleus of stria terminalis (BNSTov). Silencing of PKCδ neurons in CeA, but not in BNSTov, attenuates ARS-induced hypophagia. Experiments were carried out in mice of both sexes with n>5 per group. Conclusions A frontocortical descending PACAP projection controls PVN CRH mRNA production, to maintain hypothalamo-pituitary adrenal (HPA) axis activation, and regulate the endocrine response to stress. An ascending PACAPergic projection from eLPBn to PKCδ neurons in central amygdala regulates behavioral responses to stress. Defining two separate limbs of the acute stress response provides broader insight into the specific brain circuitry engaged by the psychogenic stress response.
WNT7B Regulates Cholangiocyte Proliferation and Function During Murine Cholestasis
Hepatology communications
Kosar, K;Cornuet, P;Singh, S;Lee, E;Liu, S;Gayden, J;Sato, T;Freyberg, Z;Arteel, G;Nejak-Bowen, K;
PMID: 34558852 | DOI: 10.1002/hep4.1784
We previously identified an up-regulation of specific Wnt proteins in the cholangiocyte compartment during cholestatic liver injury and found that mice lacking Wnt secretion from hepatocytes and cholangiocytes showed fewer proliferating cholangiocytes and high mortality in response to a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet, a murine model of primary sclerosing cholangitis. In vitro studies demonstrated that Wnt7b, one of the Wnts up-regulated during cholestasis, induces proliferation of cholangiocytes in an autocrine manner and increases secretion of proinflammatory cytokines. We hypothesized that loss of Wnt7b may exacerbate some of the complications of cholangiopathies by decreasing the ability of bile ducts to induce repair. Wnt7b-flox mice were bred with Krt19-cre mice to deplete Wnt7b expression in only cholangiocytes (CC) or with albumin-Cre mice to delete Wnt7b expression in both hepatocytes and cholangiocytes (HC + CC). These mice were placed on a DDC diet for 1 month then killed for evaluation. Contrary to our expectations, we found that mice lacking Wnt7b from CC and HC + CC compartments had improved biliary injury, decreased cellular senescence, and lesser bile acid accumulation after DDC exposure compared to controls, along with decreased expression of inflammatory cytokines. Although Wnt7b knockout (KO) resulted in fewer proliferating cholangiocytes, CC and HC + CC KO mice on a DDC diet also had more hepatocytes expressing cholangiocyte markers compared to wild-type mice on a DDC diet, indicating that Wnt7b suppression promotes hepatocyte reprogramming. Conclusion: Wnt7b induces a proproliferative proinflammatory program in cholangiocytes, and its loss is compensated for by conversion of hepatocytes to a biliary phenotype during cholestatic injury.
Topilko, T;Diaz, SL;Pacheco, CM;Verny, F;Rousseau, CV;Kirst, C;Deleuze, C;Gaspar, P;Renier, N;
PMID: 35123655 | DOI: 10.1016/j.neuron.2022.01.012
Optimizing reproductive fitness in mammalians requires behavioral adaptations during pregnancy. Maternal preparatory nesting is an essential behavior for the survival of the upcoming litter. Brain-wide immediate early gene mapping in mice evoked by nesting sequences revealed that phases of nest construction strongly activate peptidergic neurons of the Edinger-Westphal nucleus in pregnant mice. Genetic ablation, bidirectional neuromodulation, and in vitro and in vivo activity recordings demonstrated that these neurons are essential to modulate arousal before sleep to promote nesting specifically. We show that these neurons enable the behavioral effects of progesterone on preparatory nesting by modulating a broad network of downstream targets. Our study deciphers the role of midbrain CART+ neurons in behavioral adaptations during pregnancy vital for reproductive fitness.
Polinski NK, Gombash SE, Manfredsson FP, Lipton JW, Kemp CJ, Cole-Strauss A, Kanaan NM, Steece-Collier K, Kuhn NC, Wohlgenant SL, Sortwell CE.
PMID: http
Clinical trials are examining the efficacy of viral vector-mediated gene delivery for treating Parkinson’s disease (PD). While viral vector strategies have been successful in preclinical studies, to date clinical trials have disappointed. This may be due to the fact that preclinical studies fail to account for aging. Aging is the single greatest risk factor for developing PD and age alters cellular processes utilized by viral vectors. We hypothesized that the aged brain would be relatively resistant to transduction when compared to the young adult. We examined recombinant adeno-associated virus 2/5 mediated green fluorescent protein (rAAV2/5 GFP) expression in the young adult and aged rat nigrostriatal system. GFP overexpression was produced in both age groups. However, following rAAV2/5 GFP injection to the substantia nigra (SN) aged rats displayed 40-60% less GFP protein in the striatum, regardless of rat strain or duration of expression. Further, aged rats exhibited 40% fewer cells expressing GFP and 4-fold less GFP mRNA. rAAV2/5-mediated gene transfer is compromised in the aged rat midbrain, with deficiencies in early steps of transduction leading to significantly less mRNA and protein expression.
Kim, S;Oh, H;Choi, SH;Yoo, YE;Noh, YW;Cho, Y;Im, GH;Lee, C;Oh, Y;Yang, E;Kim, G;Chung, WS;Kim, H;Kang, H;Bae, Y;Kim, SG;Kim, E;
PMID: 36130507 | DOI: 10.1016/j.celrep.2022.111398
Myelin transcription factor 1 like (Myt1l), a zinc-finger transcription factor, promotes neuronal differentiation and is implicated in autism spectrum disorder (ASD) and intellectual disability. However, it remains unclear whether Myt1l promotes neuronal differentiation in vivo and its deficiency in mice leads to disease-related phenotypes. Here, we report that Myt1l-heterozygous mutant (Myt1l-HT) mice display postnatal age-differential ASD-related phenotypes: newborn Myt1l-HT mice, with strong Myt1l expression, show ASD-like transcriptomic changes involving decreased synaptic gene expression and prefrontal excitatory synaptic transmission and altered righting reflex. Juvenile Myt1l-HT mice, with markedly decreased Myt1l expression, display reverse ASD-like transcriptomes, increased prefrontal excitatory transmission, and largely normal behaviors. Adult Myt1l-HT mice show ASD-like transcriptomes involving astrocytic and microglial gene upregulation, increased prefrontal inhibitory transmission, and behavioral deficits. Therefore, Myt1l haploinsufficiency leads to ASD-related phenotypes in newborn mice, which are temporarily normalized in juveniles but re-appear in adults, pointing to continuing phenotypic changes long after a marked decrease of Myt1l expression in juveniles.
Jin, S;Maddern, XJ;Campbell, EJ;Lawrence, AJ;
PMID: 36038028 | DOI: 10.1016/j.neulet.2022.136858
Projections to the striatum are well-identified. For example, in the ventral striatum, two major inputs to the medial nucleus accumbens shell include the ventral subiculum and basolateral amygdala. However, the chemical phenotype(s) of these projection neurons remain unclear. In this study, we examined amygdalostriatal and corticostriatal connectivity in rats using injections of the retrograde tracer cholera toxin b into the nucleus accumbens shell. To determine the neurotransmitter identity of projection neurons, we combined retrograde tracing with RNAscope in-situ hybridization, using mRNA probes against vesicular transporters associated with glutamatergic (VGluT1 - Slc17a7, VGluT2 - Slc17a6) or GABAergic (VGaT - Slc32a1) neurotransmission. Confocal imaging was used to examine vesicular transporter mRNA expression in the ventral subiculum and basolateral amygdala inputs to the nucleus accumbens shell. Both projections contained mostly VGluT1-expressing neurons. Interestingly, almost a quarter of ventral subiculum to nucleus accumbens shell projections co-expressed VGluT1 and VGluT2 compared to a relatively small number (∼3%) that were co-expressed in basolateral amygdala to nucleus accumbens shell afferents. However, almost a quarter of basolateral amygdala to nucleus accumbens shell projections were VGaT-positive. These findings highlight the diverse proportions of glutamatergic and GABAergic afferents in two major projections to the nucleus accumbens shell and raise important questions for functional studies.
MOLECULAR THERAPY — METHODS & CLINICAL DEVELOPMENT
Polinski NK, Manfredsson FP, Benskey MJ, Fischer DL, Kemp CJ, Steece-Collier K, Sandoval IM, Paumier KL, Sortwell CE.
PMID: - | DOI: 10.1038/mtm.2016.82
Therapeutic protein delivery using viral vectors has shown promise in preclinical models of Parkinson’s disease (PD) but clinical trial success remains elusive. This may partially be due to a failure to include advanced age as a covariate despite aging being the primary risk factor for PD. We investigated transgene expression following intracerebral injections of recombinant adeno-associated virus pseudotypes 2/2 (rAAV2/2), 2/5 (rAAV2/5), 2/9 (rAAV2/9), and lentivirus (LV) expressing green fluorescent protein (GFP) in aged versus young adult rats. Both rAAV2/2 and rAAV2/5 yielded lower GFP expression following injection to either the aged substantia nigra or striatum. rAAV2/9-mediated GFP expression was deficient in the aged striatonigral system but displayed identical transgene expression between ages in the nigrostriatal system. Young and aged rats displayed equivalent GFP levels following LV injection to the striatonigral system but LV-delivered GFP was deficient in delivering GFP to the aged nigrostriatal system. Notably, age-related transgene expression deficiencies revealed by protein quantitation were poorly predicted by GFP-immunoreactive cell counts. Further, in situ hybridization for the viral CβA promoter revealed surprisingly limited tropism for astrocytes compared to neurons. Our results demonstrate that aging is a critical covariate to consider when designing gene therapy approaches for PD.
Rouhanifard SH, Mellis IA, Dunagin M, Bayatpour S, Jiang CL, Dardani I, Symmons O, Emert B, Torre E, Cote A, Sullivan A, Stamatoyannopoulos JA, Raj A.
PMID: 30418432 | DOI: 10.1038/nbt.4286
Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and nonspecific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400-fold) signal amplification. ClampFISH probes form a 'C' configuration upon hybridization to the sequence of interest in a double helical manner. The ends of the probes are ligated together using bio-orthogonal click chemistry, effectively locking the probes around the target. Iterative rounds of hybridization and click amplify the fluorescence intensity. We show that clampFISH enables the detection of RNA species with low-magnification microscopy and in RNA-based flow cytometry. Additionally, we show that the modular design of clampFISH probes allows multiplexing of RNA and DNA detection, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH can be applied in tissue samples.
Mathys H, Davila-Velderrain J, Peng Z, Gao F, Mohammadi S, Young JZ, Menon M, He L, Abdurrob F, Jiang X, Martorell AJ, Ransohoff RM, Hafler BP, Bennett DA, Kellis M, Tsai LH.
PMID: 31042697 | DOI: 10.1038/s41586-019-1195-2
Alzheimer's disease (AD) is a pervasive neurodegenerative disorder, the molecular and cellular complexity of which remains poorly understood. Here, we profiled and analysed 80,660 single-nucleus transcriptomes from prefrontal cortex of 48 individuals with varying degrees of AD pathology. We identified transcriptionally-distinct subpopulations across six major brain cell-types, including those associated with pathology and characterized by regulators of myelination, inflammation, and neuron survival. The strongest AD-associated changes appeared early in pathological progression and were highly cell-type-specific, whereas genes upregulated in late-stage were common across cell types and primarily involved in global stress response. Surprisingly, we found an overrepresentation of female cells in AD-associated subpopulations, and substantially different transcriptional responses between sexes in multiple cell types, including oligodendrocytes. Overall, myelination-related processes were recurrently perturbed in multiple cell types, suggesting a key role in AD pathophysiology. Our single-celltranscriptomic resource provides a first blueprint for interrogating the molecular underpinnings and cellular basis of AD.
