Herman Moreno

Lack of parvalbumin in mice leads to behavioral deficits relevant to all human autism core symptoms and related neural morphofunctional abnormalities

Gene mutations and gene copy number variants are associated with autism spectrum disorders (ASDs). Affected gene products are often part of signaling networks implicated in synapse formation and/or function leading to alterations in the excitation/inhibition (E/I) balance. Although the network of parvalbumin (PV)-expressing interneurons has gained particular attention in ASD, little is known on PV’s putative role with respect to ASD. Genetic mouse models represent powerful translational tools for studying the role of genetic and neurobiological factors underlying ASD. Here, we report that PV knockout mice (PV−/−) display behavioral phenotypes with relevance to all three core symptoms present in human ASD patients: abnormal reciprocal social interactions, impairments in communication and repetitive and stereotyped patterns of behavior. PV-depleted mice also showed several signs of ASD-associated comorbidities, such as reduced pain sensitivity and startle responses yet increased seizure susceptibility, whereas no evidence for behavioral phenotypes with relevance to anxiety, depression and schizophrenia was obtained. Reduced social interactions and communication were also observed in heterozygous (PV+/−) mice characterized by lower PV expression levels, indicating that merely a decrease in PV levels might be sufficient to elicit core ASD-like deficits. Structural magnetic resonance imaging measurements in PV−/− and PV+/− mice further revealed ASD-associated developmental neuroanatomical changes, including transient cortical hypertrophy and cerebellar hypoplasia. Electrophysiological experiments finally demonstrated that the E/I balance in these mice is altered by modification of both inhibitory and excitatory synaptic transmission. On the basis of the reported changes in PV expression patterns in several, mostly genetic rodent models of ASD, we propose that in these models downregulation of PV might represent one of the points of convergence, thus providing a common link between apparently unrelated ASD-associated synapse structure/function phenotypes.

The absence of the calcium‐buffering protein calbindin is associated with faster age‐related decline in hippocampal metabolism

Although reductions in the expression of the calcium‐buffering proteins calbindin D‐28K (CB) and parvalbumin (PV) have been observed in the aging brain, it is unknown whether these changes contribute to age‐related hippocampal dysfunction. To address this issue, we measured basal hippocampal metabolism and hippocampal structure across the lifespan of C57BL/6J, calbindin D‐28k knockout (CBKO) and parvalbumin knockout (PVKO) mice. Basal metabolism was estimated using steady state relative cerebral blood volume (rCBV), which is a variant of fMRI that provides the highest spatial resolution, optimal for the analysis of individual subregions of the hippocampal formation. We found that like primates, normal aging in C57BL/6J mice is characterized by an age‐dependent decline in rCBV‐estimated dentate gyrus (DG) metabolism. Although abnormal hippocampal fMRI signals were observed in CBKO and PVKO mice, only CBKO mice showed accelerated age‐dependent decline of rCBV‐estimated metabolism in the DG. We also found age‐independent structural changes in CBKO mice, which included an enlarged hippocampus and neocortex as well as global brain hypertrophy. These metabolic and structural changes in CBKO mice correlated with a deficit in hippocampus‐dependent learning in the active place avoidance task. Our results suggest that the decrease in CB that occurs during normal aging is involved in age‐related hippocampal metabolic decline. Our findings also illustrate the value of using multiple MRI techniques in transgenic mice to investigate mechanisms involved in the functional and structural changes that occur during aging.

Blocking effects of human tau on squid giant synapse transmission and its prevention by T-817 MA

Filamentous tau inclusions are hallmarks of Alzheimers disease and related neurodegenerative tauopathies, but the molecular mechanisms involved in tau-mediated changes in neuronal function and their possible effects on synaptic transmission are unknown. We have evaluated the effects of human tau protein injected directly into the presynaptic terminal axon of the squid giant synapse, which affords functional, structural, and biochemical analysis of its action on the synaptic release process. Indeed, we have found that at physiological concentration recombinant human tau (h-tau42) becomes phosphorylated, produces a rapid synaptic transmission block, and induces the formation of clusters of aggregated synaptic vesicles in the vicinity of the active zone. Presynaptic voltage clamp recordings demonstrate that h-tau42 does not modify the presynaptic calcium current amplitude or kinetics. Analysis of synaptic noise at the post-synaptic axon following presynaptic h-tau42 microinjection revealed an initial phase of increase spontaneous transmitter release followed by a marked reduction in noise. Finally, systemic administration of T-817MA, a proposed neuro-protective agent, rescued tau-induced synaptic abnormalities. Our results show novel mechanisms of h-tau42 mediated synaptic transmission failure and identify a potential therapeutic agent to treat tau-related neurotoxicity.

Tau pathology-mediated presynaptic dysfunction.

Brain tauopathies are characterized by abnormal processing of tau protein. While somatodendritic tau mislocalization has attracted considerable attention in tauopathies, the role of tau pathology in axonal transport, connectivity and related dysfunctions remains obscure. We have previously shown using the squid giant synapse that presynaptic microinjection of recombinant human tau protein (htau42) results in failure of synaptic transmission. Here, we evaluated molecular mechanisms mediating this effect. Thus, the initial event, observed after htau42 presynaptic injection, was an increase in transmitter release. This event was mediated by calcium release from intracellular stores and was followed by a reduction in evoked transmitter release. The effect of htau42 on synaptic transmission was recapitulated by a peptide comprising the phosphatase-activating domain of tau, suggesting activation of phosphotransferases. Accordingly, findings indicated that htau42-mediated toxicity involves the activities of both GSK3 and Cdk5 kinases.

Role of Liver X Receptor in AD Pathophysiology

Alzheimers disease (AD) is the major cause of dementia worldwide. The pharmacological activation of nuclear receptors (Liver X receptors: LXRs or Retinoid X receptors: RXR) has been shown to induce overexpression of the ATP-Binding Cassette A1 (ABCA1) and Apolipoprotein E (ApoE), changes that are associated with improvement in cognition and reduction of amyloid beta pathology in amyloidogenic AD mouse models (i.e. APP, PS1: 2tg-AD). Here we investigated whether treatment with a specific LXR agonist has a measurable impact on the cognitive impairment in an amyloid and Tau AD mouse model (3xTg-AD: 12-months-old; three months treatment). The data suggests that the LXR agonist GW3965 is associated with increased expression of ApoE and ABCA1 in the hippocampus and cerebral cortex without a detectable reduction of the amyloid load. We also report that most cells overexpressing ApoE (86±12%) are neurons localized in the granular cell layer of the hippocampus and entorhinal cortex. In the GW3965 treated 3xTg-AD mice we also observed reduction in astrogliosis and increased number of stem and proliferating cells in the subgranular zone of the dentate gyrus. Additionally, we show that GW3965 rescued hippocampus long term synaptic plasticity, which had been disrupted by oligomeric amyloid beta peptides. The effect of GW3965 on synaptic function was protein synthesis dependent. Our findings identify alternative functional/molecular mechanisms by which LXR agonists may exert their potential benefits as a therapeutic strategy against AD.

Targeting CDK5 post-stroke provides long-term neuroprotection and rescues synaptic plasticity.

Post-stroke cognitive impairment is a major cause of long-term neurological disability. The prevalence of post-stroke cognitive deficits varies between 20% and 80% depending on brain region, country, and diagnostic criteria. The biochemical mechanisms underlying post-stroke cognitive impairment are not known in detail. Cyclin-dependent kinase 5 is involved in neurodegeneration, and its dysregulation contributes to cognitive disorders and dementia. Here, we administered cyclin-dependent kinase 5-targeting gene therapy to the right hippocampus of ischemic rats after transient right middle cerebral artery occlusion. Cyclin-dependent kinase 5 RNA interference prevented the impairment of reversal learning four months after ischemia as well as neuronal loss, tauopathy, and microglial hyperreactivity. Additionally, cyclin-dependent kinase 5 silencing increased the expression of brain-derived neurotrophic factor in the hippocampus. Furthermore, deficits in hippocampal long-term potentiation produced by excitotoxic stimulation were rescued by pharmacological blockade of cyclin-dependent kinase 5. This recovery was blocked by inhibition of the TRKB receptor. In summary, these findings demonstrate the beneficial impact of cyclin-dependent kinase 5 reduction in preventing long-term post-ischemic neurodegeneration and cognitive impairment as well as the role of brain-derived neurotrophic factor/TRKB in the maintenance of normal synaptic plasticity.

Enhanced synaptic transmission at the squid giant synapse by artificial seawater based on physically modified saline

Superfusion of the squid giant synapse with artificial seawater (ASW) based on isotonic saline containing oxygen nanobubbles (RNS60 ASW) generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP) and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa++ amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5–10 min of RNS60 ASW superfusion and was maintained for the entire recording time, up to 1 h. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic transmission enhancement.

Neuronal hyperactivity due to loss of inhibitory tone in APOE4 mice lacking Alzheimer’s disease-like pathology

The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer’s disease (AD). However, the reason APOE4 is associated with increased AD risk remains a source of debate. Neuronal hyperactivity is an early phenotype in both AD mouse models and in human AD, which may play a direct role in the pathogenesis of the disease. Here, we have identified an APOE4-associated hyperactivity phenotype in the brains of aged APOE mice using four complimentary techniques—fMRI, in vitro electrophysiology, in vivo electrophysiology, and metabolomics—with the most prominent hyperactivity occurring in the entorhinal cortex. Further analysis revealed that this neuronal hyperactivity is driven by decreased background inhibition caused by reduced responsiveness of excitatory neurons to GABAergic inhibitory inputs. Given the observations of neuronal hyperactivity in prodromal AD, we propose that this APOE4-driven hyperactivity may be a causative factor driving increased risk of AD among APOE4 carriers.The APOE4 allele is the leading risk factor for late-onset Alzheimer’s disease, but how it might contribute to the disease is not clear. Here the authors show that a mouse expressing the human APOE4 allele displays hyperactivity in the entorhinal cortex due to a decreased inhibitory tone, which may in part explain accelerated Alzheimer’s pathology in APOE4 carriers.

First translational 'Think Tank' on cerebrovascular disease, cognitive impairment and dementia.

Abstract and introduction to the workshopAs the human population continues to age, an increasing number of people will exhibit significant deficits in cognitive function and dementia. It is now recognized that cerebrovascular, metabolic and neurodegenerative diseases all play major roles in the evolution of cognitive impairment and dementia. Thus with our more recent recognition of these relationships and our need to understand and more positively impact on this world health problem, “The Leo and Anne Albert Charitable Trust” (Gene Pranzo, Trustee with significant support from Susan Brogan, Meeting Planner) provided generous support for this inaugural international workshop that was held from April 13–16, 2015 at the beautiful Ritz Carlton Golf Resort in North Naples, Florida. Researchers from SUNY Downstate Medical Center, Brooklyn, NY organized the event by selecting the present group of translationally inclined preclinical, clinical and population scientists focused on cerebrovascular disease (CVD) risk and its progression to vascular cognitive impairment (VCI) and dementia. Participants at the workshop addressed important issues related to aging, cognition and dementia by: (1) sharing new data, information and perspectives that intersect vascular, metabolic and neurodegenerative diseases, (2) discussing gaps in translating population risk, clinical and preclinical information to the progression of cognitive loss, and (3) debating new approaches and methods to fill these gaps that can translate into future therapeutic interventions. Participants agreed on topics for group discussion prior to the meeting and focused on specific translational goals that included promoting better understanding of dementia mechanisms, the identification of potential therapeutic targets for intervention, and discussed/debated the potential utility of diagnostic/prognostic markers. Below summarizes the new data-presentations, concepts, novel directions and specific discussion topics addressed by this international translational team at our “First Leo and Anne Albert Charitable Trust ‘Think Tank’ VCI workshop”.

Computer modeling for pharmacological treatments for dystonia

Dystonia is a movement disorder that produces involuntary muscle contractions. Current pharmacological treatments are of limited efficacy. Dystonia, like epilepsy is a disorder involving excessive activty of motor areas including motor cortex and several causal gene mutations have been identified. In order to evaluate potential novel agents for multitarget therapy for dystonia, we have developed a computer model of cortex that includes some of the complex array of molecular interactions that, along with membrane ion channels, control cell excitability.