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1.
bioRxiv ; 2024 Jun 03.
Article in English | MEDLINE | ID: mdl-38895491

ABSTRACT

It is estimated that 1 in 36 children are affected by autism spectrum disorder (ASD) in the United States, which is nearly a twofold increase from a decade ago. Recent genetic studies have identified de novo loss-of-function (dnLoF) mutations in the Down Syndrome Cell Adhesion Molecule (DSCAM) as a strong risk factor for ASD. Previous research has shown that DSCAM ablation confers social interaction deficits and perseverative behaviors in mouse models. However, it remains unknown to what extent DSCAM underexpression captures the full range of behaviors, specifically cognitive phenotypes, presented in ASD. Here, we conducted a comprehensive cognitive behavioral phenotyping which revealed that loss of one copy of DSCAM , as in the DSCAM 2J +/- mice, displayed hyperactivity, increased anxiety, and motor coordination impairments. Additionally, hippocampal-dependent learning and memory was affected, including working memory, long-term memory, and contextual fear learning. Interestingly, implicit learning processes remained intact. Therefore, DSCAM LoF produces autistic-like behaviors that are similar to human cases of ASD. These findings further support a role for DSCAM dnLoF mutations in ASD and suggest DSCAM 2J +/- as a suitable model for ASD research. Summary Statement: Autism spectrum disorder represents a growing patient population. Loss of one copy of the DSCAM gene provides a promising mouse model that reproduces autistic-like behaviors for research and therapeutic testing.

2.
Acta Neuropathol Commun ; 12(1): 85, 2024 05 31.
Article in English | MEDLINE | ID: mdl-38822433

ABSTRACT

Here, we test whether early visual and OCT rod energy-linked biomarkers indicating pathophysiology in nicotinamide nucleotide transhydrogenase (Nnt)-null 5xFAD mice also occur in Nnt-intact 5xFAD mice and whether these biomarkers can be pharmacologically treated. Four-month-old wild-type or 5xFAD C57BL/6 substrains with either a null (B6J) Nnt or intact Nnt gene (B6NTac) and 5xFAD B6J mice treated for one month with either R-carvedilol + vehicle or only vehicle (0.01% DMSO) were studied. The contrast sensitivity (CS), external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness (a proxy for low pH-triggered water removal), profile shape of the hyperreflective band just posterior to the ELM (i.e., the mitochondrial configuration within photoreceptors per aspect ratio [MCP/AR]), and retinal laminar thickness were measured. Both wild-type substrains showed similar visual performance indices and dark-evoked ELM-RPE contraction. The lack of a light-dark change in B6NTac MCP/AR, unlike in B6J mice, is consistent with relatively greater mitochondrial efficiency. 5xFAD B6J mice, but not 5xFAD B6NTac mice, showed lower-than-WT CS. Light-adapted 5xFAD substrains both showed abnormal ELM-RPE contraction and greater-than-WT MCP/AR contraction. The inner retina and superior outer retina were thinner. Treating 5xFAD B6J mice with R-carvedilol + DMSO or DMSO alone corrected CS and ELM-RPE contraction but not supernormal MCP/AR contraction or laminar thinning. These results provide biomarker evidence for prodromal photoreceptor mitochondrial dysfunction/oxidative stress/oxidative damage, which is unrelated to visual performance, as well as the presence of the Nnt gene. This pathophysiology is druggable in 5xFAD mice.


Subject(s)
Dimethyl Sulfoxide , Mice, Inbred C57BL , Animals , Mice , Dimethyl Sulfoxide/pharmacology , Biomarkers/metabolism , Mice, Transgenic , Tomography, Optical Coherence , Retinal Rod Photoreceptor Cells/drug effects , Contrast Sensitivity/drug effects , Contrast Sensitivity/physiology , Disease Models, Animal , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/metabolism , Vision, Ocular/drug effects , Vision, Ocular/physiology
3.
Stem Cell Reports ; 18(12): 2498-2514, 2023 12 12.
Article in English | MEDLINE | ID: mdl-37995702

ABSTRACT

Brain organoid methods are complicated by multiple rosette structures and morphological variability. We have developed a human brain organoid technique that generates self-organizing, single-rosette cortical organoids (SOSR-COs) with reproducible size and structure at early timepoints. Rather than patterning a 3-dimensional embryoid body, we initiate brain organoid formation from a 2-dimensional monolayer of human pluripotent stem cells patterned with small molecules into neuroepithelium and differentiated to cells of the developing dorsal cerebral cortex. This approach recapitulates the 2D to 3D developmental transition from neural plate to neural tube. Most monolayer fragments form spheres with a single central lumen. Over time, the SOSR-COs develop appropriate progenitor and cortical laminar cell types as shown by immunocytochemistry and single-cell RNA sequencing. At early time points, this method demonstrates robust structural phenotypes after chemical teratogen exposure or when modeling a genetic neurodevelopmental disorder, and should prove useful for studies of human brain development and disease modeling.


Subject(s)
Induced Pluripotent Stem Cells , Pluripotent Stem Cells , Humans , Induced Pluripotent Stem Cells/metabolism , Brain , Cell Differentiation , Organoids
4.
Neurobiol Stress ; 27: 100581, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37928820

ABSTRACT

Emotions are characterized not only by their valence but also by whether they are stable or labile. Yet, we do not understand the molecular or circuit mechanisms that control the dynamic nature of emotional responses. We have shown that glucocorticoid receptor overexpression in the forebrain (GRov) leads to a highly reactive mouse with increased anxiety behavior coupled with greater swings in emotional responses. This phenotype is established early in development and persists into adulthood. However, the neural circuitry mediating this lifelong emotional lability remains unknown. In the present study, optogenetic stimulation in ventral dentate gyrus (vDG) of GRov mice led to a greater range and a prolonged duration of anxiety behavior. cFos expression analysis showed that the amplified behavioral response to vDG activation in GRov mice is coupled to increased neuronal activity in specific brain regions. Relative to wild type mice, GRov mice displayed glutamatergic/GABAergic activation imbalance in ventral CA1 (vCA1) and selectively increased glutamatergic activation in the basal posterior amygdaloid complex. Moreover, forebrain GR overexpression led to increased activation of molecularly distinct subpopulations of neurons within the hippocampus and the posterior basolateral amygdala (pBLA) as evident from the increased cFos co-labeling in the calbindin1+ glutamatergic neurons in vCA1 and in the DARPP-32/Ppp1r1b+ glutamatergic neurons in pBLA. We propose that a molecularly distinct hippocampal-amygdala circuit is shaped by stress early in life and tunes the dynamics of emotional responses.

5.
eNeuro ; 10(5)2023 05.
Article in English | MEDLINE | ID: mdl-37156609

ABSTRACT

The nucleus accumbens (NAc) is known for its central role in reward and motivation (Day and Carelli, 2007; Floresco, 2015; Salgado and Kaplitt, 2015). Decades of research on the cellular arrangement, density, and connectivity of the NAc have identified two main subregions known as the core and shell (Záborszky et al., 1985; Berendse and Groenewegen, 1990; Zahm and Heimer, 1990). Although anatomically and functionally different, both the NAc core and shell are mainly comprised of GABAergic projection neurons known as medium spiny neurons (MSNs) (Matamales et al., 2009). Several studies have identified key morphologic differences between core and shell MSNs (Meredith et al., 1992; Forlano and Woolley, 2010) but few studies have directly addressed how core and shell MSNs differ in their intrinsic excitability (Pennartz et al., 1992; O'Donnell and Grace, 1993). Using whole-cell patch-clamp recordings in slices prepared from naive and rewarded male rats, we found that MSNs in the NAc shell were significantly more excitable than MSNs in the NAc core in both groups. In the shell, MSNs had significantly greater input resistance, lower cell capacitance, and a greater sag. This was accompanied by a lower action potential current threshold, a greater number of action potentials, and faster firing frequency compared with core MSNs. These subregional differences in intrinsic excitability could provide a potential physiological link to the distinct anatomic characteristics of core and shell MSNs and to their distinct functional roles in reward learning (Zahm, 1999; Ito and Hayen, 2011; Saddoris et al., 2015; West and Carelli, 2016).


Subject(s)
Mental Disorders , Nucleus Accumbens , Rats , Animals , Male , Nucleus Accumbens/physiology , Medium Spiny Neurons , Action Potentials/physiology , GABAergic Neurons/physiology
6.
Aging Cell ; 22(3): e13781, 2023 03.
Article in English | MEDLINE | ID: mdl-36703244

ABSTRACT

The calcium dysregulation hypothesis of brain aging posits that an age-related increase in neuronal calcium concentration is responsible for alterations in a variety of cellular processes that ultimately result in learning and memory deficits in aged individuals. We previously generated a novel transgenic mouse line, in which expression of the L-type voltage-gated calcium, CaV 1.3, is increased by ~50% over wild-type littermates. Here, we show that, in young mice, this increase is sufficient to drive changes in neuronal physiology and cognitive function similar to those observed in aged animals. Specifically, there is an increase in the magnitude of the postburst afterhyperpolarization, a deficit in spatial learning and memory (assessed by the Morris water maze), a deficit in recognition memory (assessed in novel object recognition), and an overgeneralization of fear to novel contexts (assessed by contextual fear conditioning). While overexpression of CaV 1.3 recapitulated these key aspects of brain aging, it did not produce alterations in action potential firing rates, basal synaptic communication, or spine number/density. Taken together, these results suggest that increased expression of CaV 1.3 in the aged brain is a crucial factor that acts in concert with age-related changes in other processes to produce the full complement of structural, functional, and behavioral outcomes that are characteristic of aged animals.


Subject(s)
Calcium Channels, L-Type , Calcium , Mice , Animals , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Calcium/metabolism , Cognition/physiology , Learning , Mice, Transgenic , Maze Learning , Mice, Inbred C57BL
7.
Clin Transl Med ; 12(9): e1046, 2022 09.
Article in English | MEDLINE | ID: mdl-36101963

ABSTRACT

BACKGROUND: As the field of stem cell therapy advances, it is important to develop reliable methods to overcome host immune responses in animal models. This ensures survival of transplanted human stem cell grafts and enables predictive efficacy testing. Immunosuppressive drugs derived from clinical protocols are frequently used but are often inconsistent and associated with toxic side effects. Here, using a molecular imaging approach, we show that immunosuppression targeting costimulatory molecules CD4 and CD40L enables robust survival of human xenografts in mouse brain, as compared to conventional tacrolimus and mycophenolate mofetil. METHODS: Human neural stem cells were modified to express green fluorescent protein and firefly luciferase. Cells were implanted in the fimbria fornix of the hippocampus and viability assessed by non-invasive bioluminescent imaging. Cell survival was assessed using traditional pharmacologic immunosuppression as compared to monoclonal antibodies directed against CD4 and CD40L. This paradigm was also implemented in a transgenic Alzheimer's disease mouse model. RESULTS: Graft rejection occurs within 7 days in non-immunosuppressed mice and within 14 days in mice on a traditional regimen. The addition of dual monoclonal antibody immunosuppression extends graft survival past 7 weeks (p < .001) on initial studies. We confirm dual monoclonal antibody treatment is superior to either antibody alone (p < .001). Finally, we demonstrate robust xenograft survival at multiple cell doses up to 6 months in both C57BL/6J mice and a transgenic Alzheimer's disease model (p < .001). The dual monoclonal antibody protocol demonstrated no significant adverse effects, as determined by complete blood counts and toxicity screen. CONCLUSIONS: This study demonstrates an effective immunosuppression protocol for preclinical testing of stem cell therapies. A transition towards antibody-based strategies may be advantageous by enabling stem cell survival in preclinical studies that could inform future clinical trials.


Subject(s)
Alzheimer Disease , Neural Stem Cells , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Brain , CD40 Ligand , Humans , Immunosuppression Therapy , Mice , Mice, Inbred C57BL
8.
Allergy ; 77(2): 525-539, 2022 02.
Article in English | MEDLINE | ID: mdl-34181765

ABSTRACT

BACKGROUND: Voltage-gated calcium (Cav 1) channels contribute to T-lymphocyte activation. Cav 1.2 and Cav 1.3 channels are expressed in Th2 cells but their respective roles are unknown, which is investigated herein. METHODS: We generated mice deleted for Cav 1.2 in T cells or Cav 1.3 and analyzed TCR-driven signaling. In this line, we developed original fast calcium imaging to measure early elementary calcium events (ECE). We also tested the impact of Cav 1.2 or Cav 1.3 deletion in models of type 2 airway inflammation. Finally, we checked whether the expression of both Cav 1.2 and Cav 1.3 in T cells from asthmatic children correlates with Th2-cytokine expression. RESULTS: We demonstrated non-redundant and synergistic functions of Cav 1.2 and Cav 1.3 in Th2 cells. Indeed, the deficiency of only one channel in Th2 cells triggers TCR-driven hyporesponsiveness with weakened tyrosine phosphorylation profile, a strong decrease in initial ECE and subsequent reduction in the global calcium response. Moreover, Cav 1.3 has a particular role in calcium homeostasis. In accordance with the singular roles of Cav 1.2 and Cav 1.3 in Th2 cells, deficiency in either one of these channels was sufficient to inhibit cardinal features of type 2 airway inflammation. Furthermore, Cav 1.2 and Cav 1.3 must be co-expressed within the same CD4+ T cell to trigger allergic airway inflammation. Accordingly with the concerted roles of Cav 1.2 and Cav 1.3, the expression of both channels by activated CD4+ T cells from asthmatic children was associated with increased Th2-cytokine transcription. CONCLUSIONS: Thus, Cav 1.2 and Cav 1.3 act as a duo, and targeting only one of these channels would be efficient in allergy treatment.


Subject(s)
Asthma , Calcium Channels , Animals , Asthma/metabolism , Calcium/metabolism , Calcium Channels/metabolism , Cytokines/metabolism , Humans , Inflammation/metabolism , Mice , Receptors, Antigen, T-Cell/metabolism , Th2 Cells/metabolism
9.
Semin Thromb Hemost ; 48(3): 288-300, 2022 Apr.
Article in English | MEDLINE | ID: mdl-34942669

ABSTRACT

Tissue plasminogen activator's (tPA) fibrinolytic function in the vasculature is well-established. This specific role for tPA in the vasculature, however, contrasts with its pleiotropic activities in the central nervous system. Numerous physiological and pathological functions have been attributed to tPA in the central nervous system, including neurite outgrowth and regeneration; synaptic and spine plasticity; neurovascular coupling; neurodegeneration; microglial activation; and blood-brain barrier permeability. In addition, multiple substrates, both plasminogen-dependent and -independent, have been proposed to be responsible for tPA's action(s) in the central nervous system. This review aims to dissect a subset of these different functions and the different molecular mechanisms attributed to tPA in the context of learning and memory. We start from the original research that identified tPA as an immediate-early gene with a putative role in synaptic plasticity to what is currently known about tPA's role in a learning and memory disorder, Alzheimer's disease. We specifically focus on studies demonstrating tPA's involvement in the clearance of amyloid-ß and neurovascular coupling. In addition, given that tPA has been shown to regulate blood-brain barrier permeability, which is perturbed in Alzheimer's disease, this review also discusses tPA-mediated vascular dysfunction and possible alternative mechanisms of action for tPA in Alzheimer's disease pathology.


Subject(s)
Alzheimer Disease , Tissue Plasminogen Activator , Alzheimer Disease/drug therapy , Humans , Neuronal Plasticity
10.
J Biol Chem ; 296: 100508, 2021.
Article in English | MEDLINE | ID: mdl-33675750

ABSTRACT

The aggregation of amyloidogenic polypeptides is strongly linked to several neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. Conformational antibodies that selectively recognize protein aggregates are leading therapeutic agents for selectively neutralizing toxic aggregates, diagnostic and imaging agents for detecting disease, and biomedical reagents for elucidating disease mechanisms. Despite their importance, it is challenging to generate high-quality conformational antibodies in a systematic and site-specific manner due to the properties of protein aggregates (hydrophobic, multivalent, and heterogeneous) and limitations of immunization (uncontrolled antigen presentation and immunodominant epitopes). Toward addressing these challenges, we have developed a systematic directed evolution procedure for affinity maturing antibodies against Alzheimer's Aß fibrils and selecting variants with strict conformational and sequence specificity. We first designed a library based on a lead conformational antibody by sampling combinations of amino acids in the antigen-binding site predicted to mediate high antibody specificity. Next, we displayed this library on the surface of yeast, sorted it against Aß42 aggregates, and identified promising clones using deep sequencing. The resulting antibodies displayed similar or higher affinities than clinical-stage Aß antibodies (aducanumab and crenezumab). Moreover, the affinity-matured antibodies retained high conformational specificity for Aß aggregates, as observed for aducanumab and unlike crenezumab. Notably, the affinity-maturated antibodies displayed extremely low levels of nonspecific interactions, as observed for crenezumab and unlike aducanumab. We expect that our systematic methods for generating antibodies with unique combinations of desirable properties will improve the generation of high-quality conformational antibodies specific for diverse types of aggregated conformers.


Subject(s)
Amyloid/metabolism , Antibodies, Monoclonal/immunology , Brain/pathology , Amyloid/antagonists & inhibitors , Amyloid/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Binding Sites, Antibody , Brain/immunology , Case-Control Studies , Humans , Mice , Models, Molecular , Protein Conformation
11.
Stem Cells Transl Med ; 10(1): 83-97, 2021 01.
Article in English | MEDLINE | ID: mdl-32841522

ABSTRACT

Stem cell transplantation therapies are currently under investigation for central nervous system disorders. Although preclinical models show benefit, clinical translation is somewhat limited by the absence of reliable noninvasive methods to confirm targeting and monitor transplanted cells in vivo. Here, we assess a novel magnetic resonance imaging (MRI) contrast agent derived from magnetotactic bacteria, magneto-endosymbionts (MEs), as a translatable methodology for in vivo tracking of stem cells after intracranial transplantation. We show that ME labeling provides robust MRI contrast without impairment of cell viability or other important therapeutic features. Labeled cells were visualized immediately post-transplantation and over time by serial MRI in nonhuman primate and mouse brain. Postmortem tissue analysis confirmed on-target grft location, and linear correlations were observed between MRI signal, cell engraftment, and tissue ME levels, suggesting that MEs may be useful for determining graft survival or rejection. Overall, these findings indicate that MEs are an effective tool for in vivo tracking and monitoring of cell transplantation therapies with potential relevance to many cellular therapy applications.


Subject(s)
Bacteria , Brain , Magnetic Resonance Imaging , Magnetics , Neural Stem Cells , Animals , Brain/diagnostic imaging , Cell Tracking , Contrast Media , Humans , Mice , Primates , Rodentia , Stem Cell Transplantation
12.
J Alzheimers Dis ; 78(4): 1419-1438, 2020.
Article in English | MEDLINE | ID: mdl-33164928

ABSTRACT

BACKGROUND: In animal models and tissue preparations, calcium dyshomeostasis is a biomarker of aging and Alzheimer's disease that is associated with synaptic dysfunction, neuritic pruning, and dysregulated cellular processes. It is unclear, however, whether the onset of calcium dysregulation precedes, is concurrent with, or is the product of pathological cellular events (e.g., oxidation, amyloid-ß production, and neuroinflammation). Further, neuronal calcium dysregulation is not always present in animal models of amyloidogenesis, questioning its reliability as a disease biomarker. OBJECTIVE: Here, we directly tested for the presence of calcium dysregulation in dorsal hippocampal neurons in male and female 5×FAD mice on a C57BL/6 genetic background using sharp electrodes coupled with Oregon-green Bapta-1 imaging. We focused on three ages that coincide with the course of amyloid deposition: 1.5, 4, and 10 months old. METHODS: Outcome variables included measures of the afterhyperpolarization, short-term synaptic plasticity, and calcium kinetics during synaptic activation. Quantitative analyses of spatial learning and memory were also conducted using the Morris water maze. Main effects of sex, age, and genotype were identified on measures of electrophysiology and calcium imaging. RESULTS: Measures of resting Oregon-green Bapta-1 fluorescence showed significant reductions in the 5×FAD group compared to controls. Deficits in spatial memory, along with increases in Aß load, were detectable at older ages, allowing us to test for temporal associations with the onset of calcium dysregulation. CONCLUSION: Our results provide evidence that reduced, rather than elevated, neuronal calcium is identified in this 5×FAD model and suggests that this surprising result may be a novel biomarker of AD.


Subject(s)
Aging/metabolism , Alzheimer Disease/metabolism , Calcium/metabolism , Hippocampus/metabolism , Neurons/metabolism , Plaque, Amyloid/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/physiopathology , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Female , Hippocampus/cytology , Hippocampus/physiopathology , Humans , Male , Mice , Mice, Transgenic , Morris Water Maze Test , Neuronal Plasticity , Optical Imaging , Patch-Clamp Techniques , Plaque, Amyloid/physiopathology , Presenilin-1/genetics , Sex Factors , Spatial Learning , Spatial Memory
13.
Neurobiol Dis ; 143: 105016, 2020 09.
Article in English | MEDLINE | ID: mdl-32653673

ABSTRACT

The ubiquitin-binding proteasomal shuttle protein UBQLN2 is implicated in common neurodegenerative disorders due to its accumulation in disease-specific aggregates and, when mutated, directly causes familial frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). Like other proteins linked to FTD/ALS, UBQLN2 undergoes phase separation to form condensates. The relationship of UBQLN2 phase separation and accumulation to neurodegeneration, however, remains uncertain. Employing biochemical, neuropathological and behavioral assays, we studied the impact of overexpressing WT or mutant UBQLN2 in the CNS of transgenic mice. Expression of UBQLN2 harboring a pathogenic mutation (P506T) elicited profound and widespread intraneuronal inclusion formation and aggregation without prominent neurodegenerative or behavioral changes. Both WT and mutant UBQLN2 formed ubiquitin- and P62-positive inclusions in neurons, supporting the view that UBQLN2 is intrinsically prone to phase separate, with the size, shape and frequency of inclusions depending on expression level and the presence or absence of a pathogenic mutation. Overexpression of WT or mutant UBQLN2 resulted in a dose-dependent decrease in levels of a key interacting chaperone, HSP70, as well as dose-dependent profound degeneration of the retina. We conclude that, at least in mice, robust aggregation of a pathogenic form of UBQLN2 is insufficient to cause neuronal loss recapitulating that of human FTD/ALS. Our results nevertheless support the view that altering the normal cellular balance of UBQLN2, whether wild type or mutant protein, has deleterious effects on cells of the CNS and retina that likely reflect perturbations in ubiquitin-dependent protein homeostasis.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy-Related Proteins/metabolism , Disease Models, Animal , Nerve Degeneration/metabolism , Neurodegenerative Diseases/metabolism , Neurons/metabolism , Adaptor Proteins, Signal Transducing/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Autophagy-Related Proteins/genetics , Frontotemporal Dementia/metabolism , Frontotemporal Dementia/pathology , Mice , Mice, Inbred C57BL , Mutation , Nerve Degeneration/pathology , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/pathology , Neurons/pathology , Proteostasis/physiology
14.
Mol Psychiatry ; 25(12): 3164-3177, 2020 12.
Article in English | MEDLINE | ID: mdl-32404949

ABSTRACT

There is a paucity in the development of new mechanistic insights and therapeutic approaches for treating psychiatric disease. One of the major challenges is reflected in the growing consensus that risk for these diseases is not determined by a single gene, but rather is polygenic, arising from the action and interaction of multiple genes. Canonically, experimental models in mice have been designed to ascertain the relative contribution of a single gene to a disease by systematic manipulation (e.g., mutation or deletion) of a known candidate gene. Because these studies have been largely carried out using inbred isogenic mouse strains, in which there is no (or very little) genetic diversity among subjects, it is difficult to identify unique allelic variants, gene modifiers, and epigenetic factors that strongly affect the nature and severity of these diseases. Here, we review various methods that take advantage of existing genetic diversity or that increase genetic variance in mouse models to (1) strengthen conclusions of single-gene function; (2) model diversity among human populations; and (3) dissect complex phenotypes that arise from the actions of multiple genes.


Subject(s)
Mental Disorders , Alleles , Animals , Mental Disorders/genetics , Mice , Mice, Inbred Strains , Multifactorial Inheritance/genetics , Phenotype
15.
Neurobiol Learn Mem ; 173: 107230, 2020 09.
Article in English | MEDLINE | ID: mdl-32407963

ABSTRACT

Over the last two decades there has been significant progress towards understanding the neural substrates that underlie age-related cognitive decline. Although many of the exact molecular and cellular mechanisms have yet to be fully understood, there is consensus that alterations in neuronal calcium homeostasis contribute to age-related deficits in learning and memory. Furthermore, it is thought that the age-related changes in calcium homeostasis are driven, at least in part, by changes in calcium channel expression. In this review, we focus on the role of a specific class of calcium channels: L-type voltage-gated calcium channels (LVGCCs). We provide the reader with a general introduction to voltage-gated calcium channels, followed by a more detailed description of LVGCCs and how they serve to regulate neuronal excitability via the post burst afterhyperpolarization (AHP). We conclude by reviewing studies that link the slow component of the AHP to learning and memory, and discuss how age-related increases in LVGCC expression may underlie cognitive decline by mediating a decrease in neuronal excitability.


Subject(s)
Aging/metabolism , Brain/metabolism , Calcium Channels, L-Type/metabolism , Neurons/metabolism , Animals , Humans , Learning/physiology , Membrane Potentials/physiology , Memory/physiology
16.
Front Cell Neurosci ; 14: 122, 2020.
Article in English | MEDLINE | ID: mdl-32457579

ABSTRACT

mTORopathies are a heterogeneous group of neurological disorders characterized by malformations of cortical development (MCD), enhanced cellular mechanistic target of rapamycin (mTOR) signaling, and epilepsy that results from mutations in mTOR pathway regulatory genes. Homozygous mutations (del exon 9-13) in the pseudokinase STE20-related kinase adaptor alpha (STRAD-α; STRADA), an mTOR modulator, are associated with Pretzel Syndrome (PS), a neurodevelopmental disorder within the Old Order Mennonite Community characterized by megalencephaly, intellectual disability, and intractable epilepsy. To study the cellular mechanisms of STRADA loss, we generated CRISPR-edited Strada mouse N2a cells, a germline mouse Strada knockout (KO-/-) strain, and induced pluripotent stem cell (iPSC)-derived neurons from PS individuals harboring the STRADA founder mutation. Strada KO in vitro leads to enhanced mTOR signaling and iPSC-derived neurons from PS individuals exhibit enhanced cell size and mTOR signaling activation, as well as subtle alterations in electrical firing properties e.g., increased input resistance, a more depolarized resting membrane potential, and decreased threshold for action potential (AP) generation. Strada-/- mice exhibit high rates of perinatal mortality and out of more than 100 litters yielding both WT and heterozygous pups, only eight Strada-/- animals survived past P5. Strada-/- mice are hypotonic and tremulous. Histopathological examination (n = 5 mice) revealed normal gross brain organization and lamination but all had ventriculomegaly. Ectopic neurons were seen in all five Strada-/- brains within the subcortical white matter mirroring what is observed in human PS brain tissue. These distinct experimental platforms demonstrate that STRADA modulates mTOR signaling and is a key regulator of cell size, neuronal excitability, and cortical lamination.

17.
J Neurosci ; 39(38): 7604-7614, 2019 09 18.
Article in English | MEDLINE | ID: mdl-31270158

ABSTRACT

Dysregulated adult hippocampal neurogenesis occurs in many temporal lobe epilepsy (TLE) models. Most dentate granule cells (DGCs) generated in response to an epileptic insult develop features that promote increased excitability, including ectopic location, persistent hilar basal dendrites (HBDs), and mossy fiber sprouting. However, some appear to integrate normally and even exhibit reduced excitability compared to other DGCs. To examine the relationship between DGC birthdate, morphology, and network integration in a model of TLE, we retrovirally birthdated either early-born [EB; postnatal day (P)7] or adult-born (AB; P60) DGCs. Male rats underwent pilocarpine-induced status epilepticus (SE) or sham treatment at P56. Three to six months after SE or sham treatment, we used whole-cell patch-clamp and fluorescence microscopy to record spontaneous excitatory and inhibitory currents from birthdated DGCs. We found that both AB and EB populations of DGCs recorded from epileptic rats received increased excitatory input compared with age-matched controls. Interestingly, when AB populations were separated into normally integrated (normotopic) and aberrant (ectopic or HBD-containing) subpopulations, only the aberrant populations exhibited a relative increase in excitatory input (amplitude, frequency, and charge transfer). The ratio of excitatory-to-inhibitory input was most dramatically upregulated for ectopically localized DGCs. These data provide definitive physiological evidence that aberrant integration of post-SE, AB DGCs contributes to increased synaptic drive and support the idea that ectopic DGCs serve as putative hub cells to promote seizures.SIGNIFICANCE STATEMENT Adult dentate granule cell (DGC) neurogenesis is altered in rodent models of temporal lobe epilepsy (TLE). Some of the new neurons show abnormal morphology and integration, but whether adult-generated DGCs contribute to the development of epilepsy is controversial. We examined the synaptic inputs of age-defined populations of DGCs using electrophysiological recordings and fluorescent retroviral reporter birthdating. DGCs generated neonatally were compared with those generated in adulthood, and adult-born (AB) neurons with normal versus aberrant morphology or integration were examined. We found that AB, ectopically located DGCs exhibit the most pro-excitatory physiological changes, implicating this population in seizure generation or progression.


Subject(s)
Dentate Gyrus/cytology , Dentate Gyrus/physiology , Epilepsy, Temporal Lobe/physiopathology , Neurons/cytology , Neurons/physiology , Animals , Male , Neurogenesis/physiology , Rats , Rats, Sprague-Dawley
18.
Genes Brain Behav ; 18(7): e12575, 2019 09.
Article in English | MEDLINE | ID: mdl-30973205

ABSTRACT

Fear conditioning is an associative learning process by which organisms learn to avoid environmental stimuli that are predictive of aversive outcomes. Fear extinction learning is a process by which avoidance of fear-conditioned stimuli is attenuated when the environmental stimuli is no longer predictive of the aversive outcome. Aberrant fear conditioning and extinction learning are key elements in the development of several anxiety disorders. The 129S1 inbred strain of mice is used as an animal model for maladaptive fear learning because this strain has been shown to generalize fear to other nonaversive stimuli and is less capable of extinguishing fear responses relative to other mouse strains, such as the C57BL/6. Here we report new environmental manipulations that enhance fear and extinction learning, including the ability to discriminate between an aversively paired tone and a neutral tone, in both the 129S1 and C57BL/6 strains of mice. Specifically, we show that discontinuous ("pipped") tone stimuli significantly enhance within-session extinction learning and the discrimination between neutral and aversively paired stimuli in both strains. Furthermore, we find that extinction training in novel contexts significantly enhances the consolidation and recall of extinction learning for both strains. Cumulatively, these results underscore how environmental changes can be leveraged to ameliorate maladaptive learning in animal models and may advance cognitive and behavioral therapeutic strategies.


Subject(s)
Extinction, Psychological , Gene-Environment Interaction , Animals , Conditioning, Classical , Fear , Female , Male , Mice , Mice, Inbred C57BL
19.
Sci Rep ; 8(1): 14776, 2018 10 03.
Article in English | MEDLINE | ID: mdl-30283042

ABSTRACT

Stem cell transplantation offers a potentially transformative approach to treating neurodegenerative disorders. The safety of cellular therapies is established in multiple clinical trials, including our own in amyotrophic lateral sclerosis. To initiate similar trials in Alzheimer's disease, efficacious cell lines must be identified. Here, we completed a preclinical proof-of-concept study in the APP/PS1 murine model of Alzheimer's disease. Human neural stem cell transplantation targeted to the fimbria fornix significantly improved cognition in two hippocampal-dependent memory tasks at 4 and 16 weeks post-transplantation. While levels of synapse-related proteins and cholinergic neurons were unaffected, amyloid plaque load was significantly reduced in stem cell transplanted mice and associated with increased recruitment of activated microglia. In vitro, these same neural stem cells induced microglial activation and amyloid phagocytosis, suggesting an immunomodulatory capacity. Although long-term transplantation resulted in significant functional and pathological improvements in APP/PS1 mice, stem cells were not identified by immunohistochemistry or PCR at the study endpoint. These data suggest integration into native tissue or the idea that transient engraftment may be adequate for therapeutic efficacy, reducing the need for continued immunosuppression. Overall, our results support further preclinical development of human neural stem cells as a safe and effective therapy for Alzheimer's disease.


Subject(s)
Alzheimer Disease/therapy , Amyloid beta-Peptides/genetics , Neural Stem Cells/pathology , Stem Cell Transplantation , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Animals , Cholinergic Neurons/metabolism , Cholinergic Neurons/pathology , Disease Models, Animal , Hippocampus/metabolism , Hippocampus/pathology , Humans , Immunosuppression Therapy/methods , Memory/physiology , Mice , Mice, Transgenic , Microglia/metabolism , Microglia/pathology , Phagocytosis/genetics , Synapses/genetics , Synapses/metabolism
20.
Proc Natl Acad Sci U S A ; 115(40): E9489-E9498, 2018 10 02.
Article in English | MEDLINE | ID: mdl-30224492

ABSTRACT

Two classes of peptide-producing neurons in the arcuate nucleus (Arc) of the hypothalamus are known to exert opposing actions on feeding: the anorexigenic neurons that express proopiomelanocortin (POMC) and the orexigenic neurons that express agouti-related protein (AgRP) and neuropeptide Y (NPY). These neurons are thought to arise from a common embryonic progenitor, but our anatomical and functional understanding of the interplay of these two peptidergic systems that contribute to the control of feeding remains incomplete. The present study uses a combination of optogenetic stimulation with viral and transgenic approaches, coupled with neural activity mapping and brain transparency visualization to demonstrate the following: (i) selective activation of Arc POMC neurons inhibits food consumption rapidly in unsated animals; (ii) activation of Arc neurons arising from POMC-expressing progenitors, including POMC and a subset of AgRP neurons, triggers robust feeding behavior, even in the face of satiety signals from POMC neurons; (iii) the opposing effects on food intake are associated with distinct neuronal projection and activation patterns of adult hypothalamic POMC neurons versus Arc neurons derived from POMC-expressing lineages; and (iv) the increased food intake following the activation of orexigenic neurons derived from POMC-expressing progenitors engages an extensive neural network that involves the endogenous opioid system. Together, these findings shed further light on the dynamic balance between two peptidergic systems in the moment-to-moment regulation of feeding behavior.


Subject(s)
Agouti Signaling Protein/biosynthesis , Arcuate Nucleus of Hypothalamus/metabolism , Feeding Behavior/physiology , Neurons/metabolism , Neuropeptide Y/biosynthesis , Pro-Opiomelanocortin/biosynthesis , Signal Transduction/physiology , Agouti Signaling Protein/genetics , Animals , Arcuate Nucleus of Hypothalamus/cytology , Feeding Behavior/psychology , Mice , Mice, Transgenic , Neurons/cytology , Neuropeptide Y/genetics , Pro-Opiomelanocortin/genetics
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