Fragile X cortex is characterized by decreased parvalbumin-expressing interneurons.

Fragile X syndrome is a genetic neurodevelopmental disorder caused by a mutation of the fragile X messenger ribonucleoprotein 1 (FMR1) gene in the X chromosome. Many fragile X syndrome cases present with autism spectrum disorder and fragile X syndrome cases account for up to 5% of all autism spectrum disorder cases. The cellular composition of the fragile X syndrome cortex is not well known. We evaluated alterations in the number of Calbindin, Calretinin, and Parvalbumin expressing interneurons across 5 different cortical areas, medial prefrontal cortex (BA46), primary somatosensory cortex (BA3), primary motor cortex (BA4), superior temporal cortex (BA22), and anterior cingulate cortex (BA24) of fragile X syndrome and neurotypical brains. Compared with neurotypical cases, fragile X syndrome brains displayed a significant reduction in the number of PV+ interneurons in all areas and of CR+ interneurons in BA22 and BA3. The number of CB+ interneurons did not differ. These findings are the first to demonstrate that fragile X syndrome brains are characterized by cortical wide PV+ interneuron deficits across multiple cortical areas. These add to the idea that deficits in PV+ interneurons could disrupt the cortical balance and promote clinical deficits in fragile X syndrome patients and help to develop novel therapies for neurodevelopmental disorders like fragile X syndrome and autism spectrum disorder.

FXTAS Neuropathology Includes Widespread Reactive Astrogliosis and White Matter Specific Astrocyte Degeneration.

OBJECTIVE: Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset progressive genetic neurodegenerative disorder that occurs in FMR1 premutation carriers. The temporal, spatial, and cell-type specific patterns of neurodegeneration in the FXTAS brain remain incompletely characterized. Intranuclear inclusion bodies are the neuropathological hallmark of FXTAS, which are largest and occur most frequently in astrocytes, glial cells that maintain brain homeostasis. Here, we characterized neuropathological alterations in astrocytes in multiple regions of the FXTAS brain. METHODS: Striatal and cerebellar sections from FXTAS cases (n = 12) and controls (n = 12) were stained for the astrocyte markers glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1L1 (ALDH1L1) using immunohistochemistry. Reactive astrogliosis severity, the prevalence of GFAP+ fragments, and astrocyte density were scored. Double label immunofluorescence was utilized to detect co-localization of GFAP and cleaved caspase-3. RESULTS: FXTAS cases showed widespread reactive gliosis in both grey and white matter. GFAP staining also revealed remarkably severe astrocyte pathology in FXTAS white matter - characterized by a significant and visible reduction in astrocyte density (-38.7% in striatum and - 32.2% in cerebellum) and the widespread presence of GFAP+ fragments reminiscent of apoptotic bodies. White matter specific reductions in astrocyte density were confirmed with ALDH1L1 staining. GFAP+ astrocytes and fragments in white matter were positive for cleaved caspase-3, suggesting that apoptosis-mediated degeneration is responsible for reduced astrocyte counts. INTERPRETATION: We have established that FXTAS neuropathology includes robust degeneration of astrocytes, which is specific to white matter. Because astrocytes are essential for maintaining homeostasis within the central nervous system, a loss of astrocytes likely further exacerbates neuropathological progression of other cell types in the FXTAS brain. ANN NEUROL 2024;95:558-575.

Insight and Recommendations for Fragile X-Premutation-Associated Conditions from the Fifth International Conference on FMR1 Premutation.

The premutation of the fragile X messenger ribonucleoprotein 1 (FMR1) gene is characterized by an expansion of the CGG trinucleotide repeats (55 to 200 CGGs) in the 5' untranslated region and increased levels of FMR1 mRNA. Molecular mechanisms leading to fragile X-premutation-associated conditions (FXPAC) include cotranscriptional R-loop formations, FMR1 mRNA toxicity through both RNA gelation into nuclear foci and sequestration of various CGG-repeat-binding proteins, and the repeat-associated non-AUG (RAN)-initiated translation of potentially toxic proteins. Such molecular mechanisms contribute to subsequent consequences, including mitochondrial dysfunction and neuronal death. Clinically, premutation carriers may exhibit a wide range of symptoms and phenotypes. Any of the problems associated with the premutation can appropriately be called FXPAC. Fragile X-associated tremor/ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), and fragile X-associated neuropsychiatric disorders (FXAND) can fall under FXPAC. Understanding the molecular and clinical aspects of the premutation of the FMR1 gene is crucial for the accurate diagnosis, genetic counseling, and appropriate management of affected individuals and families. This paper summarizes all the known problems associated with the premutation and documents the presentations and discussions that occurred at the International Premutation Conference, which took place in New Zealand in 2023.

Synaptic boutons are smaller in chandelier cell cartridges in autism.

Chandelier (Ch) cells are cortical interneurons with axon terminal structures known as cartridges that synapse on the axon initial segment of excitatory pyramidal neurons. Previous studies indicate that the number of Ch cells is decreased in autism, and that GABA receptors are decreased in the Ch cell synaptic target in the prefrontal cortex. To further identify Ch cell alterations, we examined whether the length of cartridges, and the number, density, and size of Ch cell synaptic boutons, differed in the prefrontal cortex of cases with autism versus control cases. We collected samples of postmortem human prefrontal cortex (Brodmann Area (BA) 9, 46, and 47) from 20 cases with autism and 20 age- and sex-matched control cases. Ch cells were labeled using an antibody against parvalbumin, a marker that labeles soma, cartridges, and synaptic boutons. We found no significant difference in the average length of cartridges, or in the total number or density of boutons in control subjects vs. subjects with autism. However, we found a significant decrease in the size of Ch cell boutons in those with autism. The reduced size of Ch cell boutons may result in reduced inhibitory signal transmission and impact the balance of excitation to inhibition in the prefrontal cortex in autism.

Distinct patterns of GABAergic interneuron pathology in autism are associated with intellectual impairment and stereotypic behaviors.

LAY ABSTRACT: Autism spectrum disorder is a neurodevelopmental condition characterized by deficits in sociability and communication and the presence of repetitive behaviors. How specific pathological alterations of the brain contribute to the clinical profile of autism spectrum disorder remains unknown. We previously found that a specific type of inhibitory interneuron is reduced in number in the autism spectrum disorder prefrontal cortex. Here, we assessed the relationship between interneuron reduction and autism spectrum disorder symptom severity. We collected clinical records from autism spectrum disorder (n = 20) and assessed the relationship between the severity of symptoms and interneuron number. We found that the reduced number of inhibitory interneurons that we previously reported is linked to specific symptoms of autism spectrum disorder, particularly stereotypic movements and intellectual impairments.

Decreased number and increased activation state of astrocytes in gray and white matter of the prefrontal cortex in autism.

The cerebral cortex presents with alterations in the number of specific cell types in autism spectrum disorder (ASD). Astrocytes have many functions in the brain including a role in higher cognitive functions and in inflammatory brain processes. Therefore, an alteration in number, function, and/or activation state of astrocytes, could be present in ASD. We quantified astrocyte number in the gray and white matter of the prefrontal cortex-BA9, BA46, and BA47-in 15 ASD and 15 age- and sex-matched control cases. We labeled astrocytes with antibodies against the protein GFAP and S100ß, markers of astrocytes. We found a significant decrease in the number of astrocytes in the gray and white matter of all prefrontal areas of interest with both markers. We also found an increased state of activation of GFAP+ astrocytes in all areas. A reduced number of astrocytes in the cerebral cortex in ASD could lead to impaired synaptic function and disrupted connectivity. An increased astrocyte activation may indicate a chronic mild inflammatory state of the cerebral cortex in ASD. Overall, we found that astrocytes are disrupted in ASD.

Hispano-American Brain Bank on Neurodevelopmental Disorders: An initiative to promote brain banking, research, education, and outreach in the field of neurodevelopmental disorders.

Neurodevelopmental disorders (NDDs) are conditions that present with brain dysfunction due to alterations in the processes of brain development. They present with neuropsychiatric, cognitive, and motor symptoms. Autism spectrum disorder (ASD) and Fragile X syndrome (FXS) are two of the most common NDDs. Human brain tissue is a scarce resource that is obtained from postmortem donations. In the case of NDDs, specifically autism, the reduced donation rate of brains prevents researchers to investigate its pathology and fine anatomy. The Hispano-American Brain Bank of Neurodevelopmental Disorders (Banco Hispanoamericano de CErebros de trastornos del NEurodesarrollo) or CENE is a large-scale brain bank for neurodevelopmental disorders in Hispano-America and the US. CENE's objectives are to collect and distribute brains of patients with NDDS, with a focus on ASD and FXS, to perform research, promote education of future scientists, and enhance public awareness about the importance of human tissue availability for scientific research on brain function and disease. CENE has thus far established a bilingual system of nodes and teams in several American countries including California-US, Pennsylvania-US, México, Puerto Rico, Colombia, and Dominican Republic. CENE ensures that postmortem NDD samples used in research better match the world's genetic and ethnic diversity. CENE enables and expands NDD brain research worldwide, particularly with respect to ASD and FXS.

Neuronal and glial cell number is altered in a cortical layer-specific manner in autism.

LAY ABSTRACT: The cerebral cortex affected with autism spectrum disorder presents changes in the number of neurons and glia cells, possibly leading to a dysregulation of brain circuits and affecting behavior. However, little is known about cell number alteration in specific layers of the cortex in autism spectrum disorder. We found an increase in the number of neurons and a decrease in the number of astrocytes in specific layers of the prefrontal cortex in postmortem human brains from autism spectrum disorder cases. We hypothesize that this may be due to a failure in neural stem cells to shift differentiation from neurons to glial cells during prenatal brain development. These data provide key anatomical findings that contribute to the bases of autism spectrum disorder pathogenesis.

FXTAS presents with upregulation of the cytokines IL12 and TNFα.

INTRODUCTION: Fragile X Tremor and Ataxia Syndrome is a progressive neurodegenerative disorder that develops in some FMR1 premutation carriers. The objective of this study is to characterize how cytokine levels are altered in the FXTAS brain. METHODS: Fresh frozen cerebellar tissue from FXTAS cases and controls was homogenized and analyzed for 12 different cytokines using a commercially available ELISA panel. RESULTS: Relative to controls, FXTAS cases showed large and significant increases in the cytokines IL-12 and TNFα. There were large but non-significant increases in the levels of IL-2, IL-8, and IL-10 in FXTAS cases. The cytokines IL-1A, IL-1B, IL-4 IL-6, IL-17A, IFNγ, and GM-CSF were not different between FXTAS and control subjects. CONCLUSIONS: For the first time, we demonstrate an increase in the pro-inflammatory cytokines TNFα and IL-12 in the FXTAS brain, both of which are implicated in the pathogenesis of Multiple Sclerosis, another neurodegenerative disorder that predominantly consists of white matter disease.

GABAARα2 is Decreased in the Axon Initial Segment of Pyramidal Cells in Specific Areas of the Prefrontal Cortex in Autism.

Some forms of Autism Spectrum Disorder, a neurodevelopmental syndrome characterized by impaired communication and social skills as well as repetitive behaviors, are purportedly associated with dysregulation of the excitation/inhibition balance in the cerebral cortex. Through human postmortem tissue analysis, we previously found a significant decrease in the number of a gamma-aminobutyric acid (GABA)ergic interneuron subtype, the chandelier (Ch) cell, in the prefrontal cortex of subjects with autism. Ch cells exclusively target the axon initial segment (AIS) of excitatory pyramidal (Pyr) neurons, and a single Ch cell forms synapses on hundreds of Pyr cells, indicating a possible role in maintaining electrical balance. Thus, we herein investigated this crucial link between Ch and Pyr cells in the anatomy of autism neuropathology by examining GABA receptor protein expression in the Pyr cell AIS in subjects with autism. We collected tissue from the prefrontal cortex (Brodmann Areas (BA) 9, 46, and 47) of 20 subjects with autism and 20 age- and sex-matched control subjects. Immunohistochemical staining with antibodies against the GABAA receptor subunit α2 (GABAARα2) - the subunit most prevalent in the Pyr cell AIS - revealed a significantly decreased GABAARα2 protein in the Pyr cell AIS in supragranular layers of prefrontal cortical areas BA9 and BA47 in autism. Downregulated GABAARα2 protein in the Pyr cell AIS may result from decreased GABA synthesis in the prefrontal cortex of subjects with autism, and thereby contribute to an excitation/inhibition imbalance. Our findings support the potential for GABA receptor agonists asa therapeutic tool for autism.

Fragile X syndrome and associated disorders: Clinical aspects and pathology.

This review aims to assemble many years of research and clinical experience in the fields of neurodevelopment and neuroscience to present an up-to-date understanding of the clinical presentation, molecular and brain pathology associated with Fragile X syndrome, a neurodevelopmental condition that develops with the full mutation of the FMR1 gene, located in the q27.3 loci of the X chromosome, and Fragile X-associated tremor/ataxia syndrome a neurodegenerative disease experienced by aging premutation carriers of the FMR1 gene. It is important to understand that these two syndromes have a very distinct clinical and pathological presentation while sharing the same origin: the mutation of the FMR1 gene; revealing the complexity of expansion genetics.

AAV-PHP.B Administration Results in a Differential Pattern of CNS Biodistribution in Non-human Primates Compared with Mice.

The ability of recombinant adeno-associated virus (AAV) to deliver transgenes to the CNS has allowed for several advancements in the field of gene therapy to treat brain disorders. Although most AAVs do not readily cross the blood-brain barrier and transduce the CNS following peripheral administration, AAV-PHP.B has recently been shown to transduce brains of mice with higher efficiency compared with its parent serotype, AAV9, following injection into the retro-orbital sinus. Here, we extended this foundational work by comparing AAV-PHP.B transduction efficiency in wild-type C57BL/6J mice using four clinically applicable delivery strategies including two intravascular (intra-jugular vein and intra-carotid artery) and two intra-cerebral spinal fluid (CSF) routes (intra-cisterna magna and intra-lateral ventricle). We scaled up these comparisons in a larger-animal model and evaluated transduction efficiency of AAV-PHP.B in the rhesus macaque. We found widespread and largely equal CNS transduction in mice following all four injection strategies, whereas we observed a differential pattern of transduction in macaques with broad cortical and spinal cord transduction seen after intrathecal administration and only very low transduction following intravascular administration. Taken together, these results suggest that AAV-PHP.B may be a useful gene therapy vector for neurological disorders, particularly those stemming from broad cortical or spinal cord neuropathology.

Normalizing glucocorticoid levels attenuates metabolic and neuropathological symptoms in the R6/2 mouse model of huntington's disease.

Huntington's disease (HD) is a fatal genetic neurological disorder caused by a mutation in the human Huntingtin (HTT) gene. This mutation confers a toxic gain of function of the encoded mutant huntingtin (mHTT) protein, leading to widespread neuropathology including the formation of mHTT-positive inclusion bodies, gene dysregulation, reduced levels of adult dentate gyrus neurogenesis and neuron loss throughout many regions of the brain. Additionally, because HTT is ubiquitously expressed, several peripheral tissues are also affected. HD patients suffer from progressive motor, cognitive, psychiatric, and metabolic symptoms, including weight loss and skeletal muscle wasting. HD patients also show neuroendocrine changes including a robust, significant elevation in circulating levels of the glucocorticoid, cortisol. Previously, we confirmed that the R6/2 mouse model of HD exhibits elevated corticosterone (the rodent homolog of cortisol) levels and demonstrated that experimentally elevated corticosterone exacerbates R6/2 HD symptomology, resulting in severe and rapid weight loss and a shorter latency to death. Given that efficacious therapeutics are lacking for HD, here we investigated whether normalizing glucocorticoid levels could serve as a viable therapeutic approach for this disease. We tested the hypothesis that normalizing glucocorticoids to wild-type levels would ameliorate HD symptomology. Wild-type (WT) and transgenic R6/2 mice were allocated to three treatment groups: 1) adrenalectomy with normalized, WT-level corticosterone replacement (10 µg/ml), 2) adrenalectomy with high HD-level corticosterone replacement (35 µg/ml), or 3) sham surgery with no corticosterone replacement. Normalizing corticosterone to WT levels led to an improvement in metabolic rate in male R6/2 mice, as indicated by indirect calorimetry, including a reduction in oxygen consumption and normalization of respiratory exchange ratio values (p < .05 for both). Normalizing corticosterone also ameliorated brain atrophy in female R6/2 mice and skeletal muscle wasting in both male and female R6/2 mice (p < .05 for all). Female R6/2 mice given WT-level corticosterone replacement also showed a reduction in HD neuropathological markers, including a reduction in mHTT inclusion burden in the striatum, cortex, and hippocampus (p < .05 for all). This data illustrates that ameliorating glucocorticoid dysregulation leads to a significant improvement in HD symptomology in the R6/2 mouse model and suggests that cortisol-reducing therapeutics may be of value in improving HD patient quality of life.

Corticosterone dysregulation exacerbates disease progression in the R6/2 transgenic mouse model of Huntington's disease.

Huntington's disease (HD) is a genetic neurological disorder that causes severe and progressive motor, cognitive, psychiatric, and metabolic symptoms. There is a robust, significant elevation in circulating levels of the stress hormone, cortisol, in HD patients; however, the causes and consequences of this elevation are largely uncharacterized. Here, we evaluated whether elevated levels of corticosterone, the rodent homolog of cortisol, contributed to the development of symptomology in transgenic HD mice. Wild-type (WT) and transgenic R6/2 mice were given either 1) adrenalectomy with WT-level corticosterone replacement (10ng/ml), 2) adrenalectomy with high HD-level corticosterone replacement (60ng/ml), or 3) sham surgery without replacement. R6/2 mice on HD-level replacement showed severe and rapid weight loss (p<0.05) and a shorter latency to death (p<0.01) relative to the HD mice on WT-level replacement. We further evaluated basal and stress-induced levels of circulating corticosterone in R6/2 mice throughout the course of their life. We found that R6/2 transgenic HD mice display a spontaneous elevation in circulating corticosterone levels that became significant at 10weeks of age. Furthermore, we identified significant dysregulation of circadian rhythmicity of corticosterone release measured over a 24h period compared to wild-type controls. Unexpectedly, we found that R6/2 transgenic mice show a blunted corticosterone response to restraint stress, compared to wild-type mice. Together, these data provide further evidence that HPA-axis activity is abnormal in R6/2 mice, and highlight the important role that cortisol plays in HD symptom development. Our findings suggest that cortisol-reducing therapeutics may be of value in improving HD patient quality of life.

Mouse Models for Studying Depression-Like States and Antidepressant Drugs.

Depression is a common psychiatric disorder, with diverse symptoms and high comorbidity with other brain dysfunctions. Due to this complexity, little is known about the neural and genetic mechanisms involved in depression pathogenesis. In a large proportion of patients, current antidepressant treatments are often ineffective and/or have undesirable side effects, fueling the search for more effective drugs. Animal models mimicking various symptoms of depression are indispensable in studying the biological mechanisms of this disease. Here, we summarize several popular methods for assessing depression-like symptoms in mice, and their utility in screening antidepressant drugs.

Experimental Models of Anxiety for Drug Discovery and Brain Research.

Animal models have been vital to recent advances in experimental neuroscience, including the modeling of common human brain disorders such as anxiety, depression, and schizophrenia. As mice express robust anxiety-like behaviors when exposed to stressors (e.g., novelty, bright light, or social confrontation), these phenotypes have clear utility in testing the effects of psychotropic drugs. Of specific interest is the extent to which mouse models can be used for the screening of new anxiolytic drugs and verification of their possible applications in humans. To address this problem, the present chapter will review different experimental models of mouse anxiety and discuss their utility for testing anxiolytic and anxiogenic drugs. Detailed protocols will be provided for these paradigms, and possible confounds will be addressed accordingly.

Intravascular AAV9 Administration for Delivering RNA Silencing Constructs to the CNS and Periphery.

Viral vector delivery of RNA silencing constructs, when administered into vasculature, typically results in poor central nervous system (CNS) transduction due to the inability of the vector to cross the blood-brain barrier (BBB). However, adeno-associated virus serotype 9 (AAV9) has the ability to cross the BBB and robustly transduce brain parenchyma and peripheral tissues at biologically meaningful levels when injected intravenously. Recent work by our lab has shown that this method can be used to deliver RNA silencing constructs, resulting in significant reductions in gene expression in multiple brain regions and in peripheral tissues. Here, we outline a method for delivery of AAV9 vectors expressing RNA interference (RNAi) constructs that lead to robust simultaneous transduction of mouse peripheral tissues and the CNS following a single injection into the jugular vein. Additionally, we outline methods for necropsy and immunofluorescence to detect AAV9 transduction patterns in the rodent CNS following a vascular delivery.

Single nucleotide seed modification restores in vivo tolerability of a toxic artificial miRNA sequence in the mouse brain.

Huntington's disease is a fatal neurodegenerative disease caused by polyglutamine-expansion in huntingtin (HTT). Recent work showed that gene silencing approaches, including RNA interference (RNAi), improve disease readouts in mice. To advance RNAi to the clinic, we designed miHDS1, with robust knockdown of human HTT and minimized silencing of unintended transcripts. In Rhesus macaque, AAV delivery of miHDS1 to the putamen reduced HTT expression with no adverse effects on neurological status including fine and gross motor skills, no immune activation and no induction of neuropathology out to 6 weeks post injection. Others showed safety of a different HTT-targeting RNAi in monkeys for 6 months. Application of miHDS1 to Huntington's patients requires further safety testing in normal rodents, despite the fact that it was optimized for humans. To satisfy this regulatory requirement, we evaluated normal mice after AAV.miHDS1 injection. In contrast to monkeys, neurological deficits occurred acutely in mice brain and was attributed to off-target silencing through interactions of miHDS1 with the 3'UTR of other transcripts. While we resolved miHDS1 toxicity in mouse brain and maintained miHDS1-silencing efficacy, these studies highlight that optimizing nucleic acid-based medicines for safety in humans presents challenges for safety testing in rodents or other distantly related species.

Drinking alcohol has sex-dependent effects on pair bond formation in prairie voles.

Alcohol use and abuse profoundly influences a variety of behaviors, including social interactions. In some cases, it erodes social relationships; in others, it facilitates sociality. Here, we show that voluntary alcohol consumption can inhibit male partner preference (PP) formation (a laboratory proxy for pair bonding) in socially monogamous prairie voles (Microtus ochrogaster). Conversely, female PP is not inhibited, and may be facilitated by alcohol. Behavior and neurochemical analysis suggests that the effects of alcohol on social bonding are mediated by neural mechanisms regulating pair bond formation and not alcohol's effects on mating, locomotor, or aggressive behaviors. Several neuropeptide systems involved in the regulation of social behavior (especially neuropeptide Y and corticotropin-releasing factor) are modulated by alcohol drinking during cohabitation. These findings provide the first evidence to our knowledge that alcohol has a direct impact on the neural systems involved in social bonding in a sex-specific manner, providing an opportunity to explore the mechanisms by which alcohol affects social relationships.

Intrajugular vein delivery of AAV9-RNAi prevents neuropathological changes and weight loss in Huntington's disease mice.

Huntington's disease (HD) is a fatal neurological disorder caused by a CAG repeat expansion in the HTT gene, which encodes a mutant huntingtin protein (mHTT). The mutation confers a toxic gain of function on huntingtin, leading to widespread neurodegeneration and inclusion formation in many brain regions. Although the hallmark symptom of HD is hyperkinesia stemming from striatal degeneration, several other brain regions are affected which cause psychiatric, cognitive, and metabolic symptoms. Additionally, mHTT expression in peripheral tissue is associated with skeletal muscle atrophy, cardiac failure, weight loss, and diabetes. We, and others, have demonstrated a prevention of motor symptoms in HD mice following direct striatal injection of adeno-associated viral vector (AAV) serotype 1 encoding an RNA interference (RNAi) construct targeting mutant HTT mRNA (mHTT). Here, we expand these efforts and demonstrate that an intrajugular vein injection of AAV serotype 9 (AAV9) expressing a mutant HTT-specific RNAi construct significantly reduced mHTT expression in multiple brain regions and peripheral tissues affected in HD. Correspondingly, this approach prevented atrophy and inclusion formation in key brain regions as well as the severe weight loss germane to HD transgenic mice. These results demonstrate that systemic delivery of AAV9-RNAi may provide more widespread clinical benefit for patients suffering from HD.