Autism spectrum disorder (ASD) is a complex group of individually rare neurological conditions with genetic and non-genetic causes. Despite the strong genetic component of ASD, it has been very difficult to identify the underlying disease genes. The major obstacles to gene discovery in ASD are the extreme diversity in the clinical symptoms of patients as well as the complex genetic makeup of the disorder. Next-generation sequencing technologies have allowed for recent strides in the genetics of ASD, while the molecular mechanisms underlying disease pathogenesis have remained elusive hindering therapy development. The field has been heavily focused on the role of spontaneous genetic changes that are not inherited from parents. However, these spontaneous mutations account for only ~30% of ASD cases, and cannot explain why ASD is highly heritable in humans. My talk will provide an overview of current areas of emphasis in ASD research spanning genomics and genetics, focusing on how we can leverage shared ancestry to identify recessively inherited ASD mutations. I will also discuss one specific example of a neurodevelopmental disease gene with recessive mutations, UBE3B, that functions in protein turnover and the molecular mechanisms underlying disease in this example.

Cortical plasticity is the neural mechanism by which the cerebrum adapts itself to its environment, while at the same time making it vulnerable to impoverished sensory or developmental experiences.  Like the visual system, auditory development passes through a series of sensitive periods in which circuits and connections are established and then refined by experience.  During these periods, the functional maturation of auditory processing and perception is critically dependent on adequate auditory experience.  Unfortunately, in cases of sensory deprivation, such as congenital deafness, this process is arrested.  Fortunately, it appears that this situation can be reversed in individuals that receive cochlear prosthetics.

Current research is expanding our understanding of cerebral processing and organization in the deaf.  In the congenitally deaf, higher-order areas of "deaf" auditory cortex demonstrate significant crossmodal plasticity with neurons responding to visual and somatosensory stimuli.  This crucial cerebral function results in adaptive, compensatory plasticity.  Not only can the remaining inputs reorganize to substitute for those lost, but this additional circuitry also confers enhanced abilities to the remaining systems.  In this presentation, we will review our present understanding of the structure and function of “deaf” auditory cortex using psychophysical, electrophysiological, and connectional anatomy approaches and consider how this knowledge informs our expectations of the capabilities of cochlear implants in the developing brain.

Determining the copy number of SMN1 and SMN2 genes is notoriously difficult due to the high level of homology between the two genes. These genes, that are associated with spinal muscular atrophy (SMA), only differ by a single nucleotide, which requires any copy number solution to have very high assay specificity.

For researchers focused on Alzheimer’s disease, identifying their samples as APOE allele type E2, E3, or E4 is of the utmost importance. Testing samples for APOE status can build a cohort that separates the low-risk E2 allele from the high-risk E4 allele. 

The Novallele™ copy number and genotyping assays are designed to give researchers a simple method to investigate difficult-to-analyze genetic changes. The Novallele assays generate accurate data using a simple protocol that returns results in around an hour. During the webinar, we will discuss the protocol in more detail, including scientific principles, assay workflows, and data analysis.

Machine learning approaches to pattern discovery in protein-protein interaction networks of Huntington’s disease brain and model organisms are revealing novel connections of the disease state to the processes of DNA and chromosome damage-repair pathways. Some of these computational inferences have been tested and validated in Huntington’s disease experimental models, suggesting provocative therapeutic target concepts.

In consciousness studies, a longstanding controversy concerns whether activity in the prefrontal cortical (PFC) region of the brain is necessary to evoke conscious experiences. Similarly, there is contrasting evidence on whether subjective confidence directly reflects sensory evidence or may depend on a late-stage estimation, related to consciousness but dissociable from sensory processes. As of yet, in humans, experimental tools have lacked the power to resolve these issues convincingly. 

We overcome this difficulty by capitalizing on the recently developed method of decoded neurofeedback (DecNef), where the occurrence of distinct neural events (e.g., spatial activation patterns) is selectively rewarded. This closed-loop training thus has the power of reinforcing purely content-specific processes that typically lie below consciousness. 

In a series of recent studies, we employed DecNef to directly reinforce neural activation patterns in areas related to representations at different levels of complexity, from simpler (e.g. orientation in visual cortex) to more composite (e.g. confidence in PFC). In all cases, the manipulations resulted in clear behavioral or physiological changes. Nevertheless, during the training sessions, participants were never conscious about the content of these localized recurring activation patterns.

This raises a very interesting point: it is likely that consciousness requires more than just a local representation, however well defined. As already proposed, consciousness may rely on concomitant activations across frontoparietal networks. Frontal cortices could thus play a crucial role in bringing content to consciousness, by virtue of being also implicated in higher order representations. Approaches combining machine learning techniques with brain imaging and closed-loop training such as DecNef may offer a strong paradigm to further explore and understand consciousness and its real neural basis.

The study of rare, neurological disorder and the development of effect treatments can pose many unique challenges. A paucity of scientists working on these disorders often limits forward progress on these disease and restricts access to good animals models, patient cells lines, and other tools for studying these disorders and can significantly hinder the speed at which treatments are developed for these disorders. In the last year, a number of groups have started to focus on how to transform "transdisciplinary" research in rare diseases to increase data sharing and expedite therapeutic development.  In this talk, we will use our work on a  rare, genetic, lysosomal storage disorder called CLN6-Batten disease as a case example of how to tackle rare disease research in a more efficient manner.  CLN6- Batten disease presents with gradual declines in motor, visual, and cognitive ability and early death by 12-15 years of age and currently no cures are available for this disease. CLN6-Batten disease is caused by mutations causing absence or reduced abundance of the CLN6 protein, making gene therapy a promising therapeutic strategy. We developed a scAAV9 vector expressing the human CLN6 (hCLN6) gene under the control of a chicken β-actin (CB) promoter. A single, postnatal intracerebroventricular (ICV) injection of scAAV9.CB.CLN6 at postnatal day 1 induced widespread, long-term expression of hCLN6 RNA and protein in the brain of Cln6nclf mice. This one-time treatment prevented or drastically reduced all of the pathological and behavioral hallmarks of Batten disease. Importantly, while all untreated CLN6 mice died between 12-14 months of age, scAAV9.CB.CLN6 significantly extended the median survival beyond 22 months of age and all of the histopathological, behavioral and cognitive parameters continued to improve throughout the lifespan of the treated mice. To our knowledge, this is the longest survival extension reported in this mouse model to date.

The inability of neurons to regenerate damaged axons within the CNS has dire consequences for victims of traumatic or ischemic brain injury and multiple neurodegenerative diseases. Like other CNS pathways, the optic nerve cannot regenerate if injured and soon the retinal ganglion cells (RGCs), the projection neurons of the eye, begin to die, precluding visual recovery. Our lab and others have identified methods that enable some RGCs to regenerate axons from the eye to the brain. Some of these methods include controlled intraocular inflammation and elevation of on-comodulin and other growth factors, deletion of genes that suppress regeneration such as pten, socs3, klf4 and -9, and counteracting cell-extrinsic suppressors of axon growth by deleting re-ceptors for Nogo and other molecules{Benowitz, 2017 #5372}. Nonetheless, even under the best of circumstances, most RGCs go on to die and only a small fraction of the surviving RGCs re-generate their axons {de Lima, 2012 #4249}. These findings imply the existence of other major suppressors of RGC survival and axon regeneration. We recently identified mobile zinc (Zn2+) one such factor. Within an hour after optic nerve injury, Zn2+ increases dramatically in synaptic vesicles of amacrine cells (ACs), the inhibitory interneurons of the retina, and is then exocytosed and accumulates in injured RGCs. Zn2+ chelation leads to the persistent survival of many RGCs and to appreciable axon regeneration, with a therapeutic window of several days{Li, 2017 #5363}. Zn2+ elevation is induced by nitric oxide (NO), a gaseous signal that is generated in a small population of ACs by the enzyme NO synthase-1 (NOS1). A novel fluorescent NO sensor reveal a dramatic increase in retinal NO levels within an hour of optic nerve damage. NO or a derivative is likely to liberate Zn2+ from metallothioneins. NO also has a second, positive effect on optic nerve regeneration through a cGMP-dependent pathway. Thus, NO generated by NOS1 in a small population of ACs is responsible for the deleterious elevation of Zn2+ after optic nerve regeneration, but also exerts a positive effect on optic nerve regeneration via cGMP sig-naling. These findings, together with others in the field, are bringing us closer to improving out-come after injury to the optic nerve and are likely to have relevance to parts of the CNS.

Monitoring protein biomarkers in cerebrospinal fluid (CSF) of patients with Alzheimer’s Disease (AD) has been highly beneficial to understanding disease progression.  While several CSF biomarkers can reproducibly distinguish normal and diseased samples, CSF is a difficult biological fluid to obtain in research studies.  The need for blood-based biomarkers of AD has driven a continuous search for novel candidates.  Here we report the development of a multiplex immunoassay to quantitatively measure seven proteins present in both CSF and blood that are involved in neurological disease:  Neurogranin, TREM2, ApoE4, FABP3, Ferritin, angiogenin, and prion protein.  Notably, presence of the APOE4 allele is prominently associated with an increased risk for AD, in comparison to the APOE2 and APOE3 alleles.  Although these ApoE isoforms differ at only one or two amino acids, our assay distinguished ApoE4 with minimal cross-reactivity. Using this novel immunoassay, we measured these 7 biomarkers in CSF, plasma, and serum from AD patients and healthy controls.  Additionally, we developed an ultrasensitive Single Molecule Counting (SMC) assay for amyloid beta 1-42 (Aβ42).  This kit could detect the Aβ42 peptide in CSF and plasma at sub-pg/mL concentrations.  This study demonstrates the value of evaluating both novel and established biomarkers of neurodegeneration across distinct sample types.

General anesthesia is a drug-induced, reversible condition comprised of five behavioral states: unconsciousness, amnesia (loss of memory), analgesia (loss of pain sensation), akinesia (immobility), and hemodynamic stability with control of the stress response. Our work shows that a primary mechanism through which anesthetics create these altered states of arousal is by initiating and maintaining highly structured oscillations. These oscillations impair communication among brain regions. We illustrate these impressive dynamics by presenting findings from our human studies of general anesthesia using high-density EEG recordings and intracranial recordings. These studies have allowed us to give a detailed characterization of the neurophysiology of loss and recovery of consciousness due to propofol. We show how the oscillatory dynamics change systematically with different anesthetic classes and with age. The age-related changes reflect brain development in children and brain aging in adults. Finally, we demonstrate that the state of general anesthesia can be rapidly reversed by activating specific brain circuits. The success of our research has depended critically on tight coupling of experiments, signal processing research and mathematical modeling. These new insights into the mechanisms of anesthetic action suggest new strategies for using the EEG to monitor the brain states of patients receiving general anesthesia, more principled strategies to dose anesthetics and new approaches to using studies of general anesthesia to gain new, fundamental insights into how the brain works. 

Reinforcement learning is a critical process though to involve the cortex, striatum, and dopaminergic circuitry.  Work from our group has found that the caudate (Cd) and dorso-lateral prefrontal cortex (DLPFC) encode unexpectedly positive outcomes, consistent with a reward prediction error.  In addition, neurons in both structures encode unexpectedly negative outcomes, which is not yet well-explained.  Moreover, this process is amenable to modulation.  Experiments in rodents, primates, and humans have consistently demonstrated that it is possible to enhance learning with timed stimulation in the Cd.  In animal models of brain injury, this stimulation resulted in accelerated recovery.   Many clinical entities can be conceptualized as disorders of this process, whether it is reduced ability to learn new associations or the inability to replace useless or maladaptive associations such as tics or compulsions. The broader implication is that timed stimulation could be clinically useful in treating disorders beyond brain injury.

How does a low-level sensory representation rise into conscious awareness? What determines whether higher-order cortical areas have access to the representational content in sensory regions? Although much evidence suggests prefrontal cortical regions are critically responsible for awareness, assessing trial-by-trial connectivity from prefrontal to lower-level sensory areas via fMRI is challenging with conventional approaches. In this talk I will present a new dynamic functional connectivity metric, based on information theory, that we are developing. The metric relies on 40-way multinomial sparse logistic regression decoding, capitalizing on a 38-subject database for which voxel-based representational patterns have been functionally aligned via Procrustean transformations. This metric may be used to assess trial-by-trial functional connectivity between prefrontal and low-level sensory areas in real-time fMRI contexts, thus providing a tool for rigorously testing the role of prefrontal cortex in conscious awareness. 

Some of the most significant hurdles faced by neuroscientists in the field of neurodegenerative disease research when seeking to acquire genetic information include sample accessibility, reproducibility, efficiency and ease of data analysis. As a proven leader in oncology research and diagnostics, NanoString technology is well-positioned to address the challenges faced by neurodegenerative disease researchers. Because NanoString does not require amplification or the use of enzymes, challenging samples like post-mortem formalin-fixed paraffin embedded (FFPE) tissue can be reliably analyzed to yield reproducible results. Moreover, NanoString offers solutions for single cell and low input samples aimed at getting the most biological information from the smallest sample, including analyte multiplexing. In contrast to other technologies, simple read-outs and data analysis put the results back in the researcher’s hand and serve to expedite the time from experimental design to discovery.  

In this webinar, we will demonstrate the robust performance of the nCounter platform for gene expression profiling of human post-mortem brain specimens, human blood samples and tissue from mouse models of neurodegenerative disease with an emphasis on performance and reproducibility in FFPE samples. Moreover, we will discuss data generated using post-mortem Alzheimer’s and Parkinson’s disease brain tissue that was profiled on our new Neuropathology gene expression panel, which includes 770 curated RNA transcripts. We will demonstrate that this panel allows for a broad and comprehensive assessment of relevant pathways and processes in neurodegenerative disease and explore important biological insights revealed by analyzing these precious samples on the nCounter platform.

Clathrin mediated endocytosis (CME) at pre-synaptic terminals is functionally coupled to neuro-transmitter release. Like other cytosolic proteins, clathrin is synthesized in cell-bodies and conveyed into axons and synapses via slow axonal transport. Although the ‘triskelial’ structural conformation and endocytic function of clathrin at synapses is well-established, the cargo-structure or transport behavior of clathrin molecules moving in slow axonal transport is virtually unknown. We defined the cargo-composition and transport-kinetics of clathrin assemblies in axons using a combination of live-cell imaging, mass-spectrometry and correlative electron microscopy in primary hippocampal cultures and neurons in-vivo. We found that axonal clathrin is transported as a unique, stable cargo-complex that is distinct from the known ephemeral triskelial structures that exist at synapses and dendrites. While its transport-kinetics resembles other slow-component cargoes, surprisingly, the transport-complex contains both clathrin assembly and disassembly proteins and is ultra structurally distinct from any known clathrin assemblage. Further, we demonstrate that the stable transported clathrin cargo complex is deposited at en passant boutons along axons. Our studies thus uncover a previously-unknown form of clathrin and clarify mechanistic details of its slow transport into axons.

Using Hebbian as well as homeostatic models of brain plasticity, the effects of neurofeedback (NFB) are examined from the theoretical perspective of EEG normalization. Within this framework, hyper- and hypo-synchronous oscillations characteristic of abnormal brain dynamics are reversed with NFB training, as seen in several brain disorders including attention-deficit hyperactivity (ADHD) and post-traumatic stress disorder (PTSD). The central thesis put forward is that NFB re-tunes pathological oscillations toward intermediate levels of coupling, which may support self-organized criticality and provide an optimal balance between network flexibility/stability.

CRISPR Cas9 nucleases have revolutionized gene editing enabling unprecedented efficiency of targeted mutagenesis.   Even with such powerful technology at hand, sophisticated projects, such as those that involve editing of neural cell types, may be challenging and time-consuming.  Similarly, due to the time and expense of exploring mechanisms of neural function neural disease modeling requires a high level of up-front design confidence.  As CRISPR becomes a focus of the molecular biology research community, MilliporeSigma seeks to share methods learned and strategies applied in our years of gene editing experience.   Today’s presentation will focus on practical Zinc Finger Nuclease and CRISPR approaches for pristine gene editing to achieve both knockout and specific sequence changes.  Special attention will be paid to publications detailing gene tagging and in vivo applications of lentiviral CRISPR reagents.

Is sensory awareness facilitated by activity in only sensory cortices? For several decades, this has been the dominant viewpoint among many prominent voices in both neuroscience and philosophy.  In this talk, I will review recent work which shows how many of the components of perceptual decision-making, including confidence, integration, and content, are all at least partially facilitated by brain regions outside early sensory areas.  Drawing upon results from psychophysical, computational, neuroimaging and neurophysiological work, I will show that far from being irrelevant for perceptual experiences, frontal regions of the brain actually contain rich representations of perceptual content, and that decision-related processes and circuitry play a key role in metacognitive judgments and sensory awareness.  These new results shed light on current debates about the locus of conscious perceptual experience, and open exciting new possibilities for probing this question going forward.

Since its initial description more than 70 years ago, autism spectrum disorder (ASD) has been diagnosed more frequently in boys. However, we remain unsure of why males are affected in greater numbers. Genetic and hormonal causes have been proposed, but clinicians and researchers are still debating whether males are more susceptible or females are more resilient. One primary hurdle in testing different hypotheses was the lack of appropriate animal models. In developing a novel mouse model of ASD and intellectual disability (ID) lacking Coiled-coil and C2 domain containing 1a, CC2D1A, which is mutated in humans with ID, ASD and seizures, we found that male mice are more severely affected than females. While males show an array of memory and sociability deficits with hyperactivity and anxiety, females only display a subset of learning impairments. In parallel, sex-specific behavioral differences correlate with male-specific reduction in activation of the transcription factor CREB in the hippocampus of KO animals. CREB function is critical for spatial memory formation, and modulation of CREB signaling rescues both molecular and behavioral deficits in male KO mice. Our results indicate that CC2D1A may establish male-specific signaling processes controlling memory. Defining how male-specific signaling is established will provide key insight on sex specific signaling in the brain, and will also lead to significant advances in the understanding of mechanisms that could lead to male bias in neurodevelopmental disorders.

Most neuropsychiatric diseases involve multifactorial systems characterized by complex interactions among genetic predisposition/resiliency, environmental/social determinants, molecular sequelae and brain network organization. The allostatic load affects remodeling of brain architecture and physiological processes in response to stressors. The epigenetic allostasis emphasizes the influence of early-life experiences on the development of neuropsychiatric pathology in susceptible individuals. This presentation will cover the neurobiological frameworks underlying three main components of allostatic load – oxidative stress, inflammation and insulin resistance (IR) while accounting for genetic (familial) and epigenetic (early-life trauma) in human models. 

Biomedical research depends on the use of model systems to explore basic biology, probe disease mechanisms, and conduct drug discovery and development. However, results from such systems are often not recapitulated in man. This observation has led us to adopt the principle that a biomedical researcher should select a model system with the greatest physiological relevance to human physiology (or pathophysiology) that is technical and economically feasible for a given project. For early stage neuroscience and CNS drug discovery studies, human neurons derived from induced pluripotent stem cells (iPSCs) can meet this need. They can replace animal cells and immortalized human cell lines, which have limited predictive power to drive research in the right direction. This presentation will provide an overview of the technology used to create iPSC-derived neurons and describe some of the characteristics of these neurons. We will also discuss examples of how these neurons can be used in research applications, with a particular emphasis on drug discovery.

The cerebellum is incorporated into the distributed neural circuits subserving motor control, cognitive processing and the modulation of emotion. This lecture provides an overview of anatomical studies in monkey and functional magnetic resonance imaging studies in humans demonstrating topographic arrangement in the cerebellum of motor and non-motor domains.  We consider motor impairments such as ataxia, dysmetria and dysarthria as resulting from lesions of the motor cerebellum predominantly in the anterior lobe, whereas executive, linguistic, spatial, and emotional impairments (the cerebellar cognitive affective syndrome, CCAS) arise following lesions of the cognitive cerebellum in the posterior lobe. Affective dysregulation occurs particularly when lesions involve the cerebellar vermis. These higher order impairments represent subcortical disconnection syndromes, manifestations of dysmetria of thought resulting from loss of the universal cerebellar transform, the unique computation that the cerebellum contributes to functionally specific subcircuits within distributed cortico-subcortical networks. Patient studies reveal that the affective component of the CCAS manifests as deficits in the domains of attentional control, emotional control, social skill set, autism spectrum disorders and psychosis spectrum disorders. This new appreciation of cerebellar circuits, functions, and deficits has relevance for understanding and treating adults and children with cerebellar disorders; investigating neuropsychiatric diseases including autism and schizophrenia; and for new therapeutic possibilities in neuropsychiatry. This is exemplified by preliminary  evidence that transcranial magnetic stimulation applied to the cerebellar vermis improves negative symptoms and enhances quality of life in patients with schizophrenia.

Objective for this lecture are:

• To define the essential elements of the distributed neural circuits linking cerebellum with association and paralimbic areas as well as sensorimotor regions of the cerebral cortex.
• Describe the clinical features and broader relevance of the cerebellar cognitive affective syndrome and the neuropsychiatry of the cerebellum.
• Explain how the dysmetria of thought theory and the concept of the universal cerebellar transform account for the cerebellar role in cognition and emotion, and the relevance this has for novel approaches to treatment of neuropsychiatric disorders.

Complex animal behavior is produced by dynamic interactions between discrete regions of the brain. As such, defining functional connections between brain regions is critical in gaining a full understanding of how the brain generates behavior. Evidence suggests that discrete regions of the cerebellar cortex functionally project to the forebrain, mediating long-range communication potentially important in motor and non-motor behaviors. However, the connectivity map remains largely incomplete owing to the challenge of driving both reliable and selective output from the cerebellar cortex, as well as the need for methods to detect region specific activation across the entire forebrain. I will discuss a combined optogenetic and fMRI (ofMRI) approach to elucidate the downstream forebrain regions modulated by activating a region of the cerebellum that induces stereotypical, ipsilateral forelimb movements. In this talk I will demonstrate that a single discrete region of the cerebellar cortex is capable of influencing motor output and the activity of a number of downstream forebrain as well as midbrain regions thought to be involved in different aspects of behavior.

Psychiatric diagnosis is inherently difficult, due to the lack of clear biomarkers or any other objective assessment. Although quantitative, the psychometric scales employed during the psychiatric interview are subjective, so psychiatrists are required to undergo extensive training before they are properly qualified to diagnose. In search of objective, quantitative insight into the structure of psychotic speech, we applied standard techniques in the field of complex networks to measure speech graph attributes in chronic patients with schizophrenia, bipolar disorder type I, and non-psychotic controls as they reported waking and dream contents. Speech graphs are simply directed graphs of word trajectories, upon which graph-theoretical measures can be applied. We show that the three different groups can be sorted just by looking at a handful of speech graph attributes, after discarding all semantic content. The technique can also be successfully extended to patients during their first clinical contact, objectively classifying diagnosis 6 months in advance and providing a diagnostic-independent “disorganization index” for speech. The results demonstrate the feasibility of the differential diagnosis of psychosis based on the analysis of speech graphs, pointing to a fast, low-cost and language-invariant tool for psychiatric diagnosis and the objective search for biomarkers. 

98% of small molecules and 100% of large molecules do not cross the blood-brain barrier (BBB). Biologics, including therapeutic antibodies (e.g. anti-amyloid antibodies), have been at the forefront of Alzheimer’s disease (AD) research, however have had limited success so far. Biologics are large molecules with limited permeability across the BBB, and in the absence of a BBB-targeting approach to deliver these biologics to the brain, the true potential of the biologic cannot be determined. One approach to deliver a biologic, including anti-amyloid antibodies, decoy receptors and neurotrophins, for AD is to engineer bispecific fusion proteins, such that one domain of the fusion protein is the therapeutic biologic of interest, and the other domain acts as a molecular Trojan horse to ferry the biologic into the brain. This talk will focus on some of our work involving the targeting of the BBB transferrin receptor to deliver biologics (e.g. anti-amyloid antibody and biologic TNF-a inhibitor) in mouse models of AD.