Identification of gene fusions associated with amyotrophic lateral sclerosis.

INTRODUCTION/AIMS: Genetics is an important risk factor for amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons. Recent findings demonstrate that in addition to specific genetic mutations, structural variants caused by genetic instability can also play a causative role in ALS. Genomic instability can lead to deletions, duplications, insertions, inversions, and translocations in the genome, and these changes can sometimes lead to fusion of distinct genes into a single transcript. Gene fusion events have been studied extensively in cancer; however, they have not been thoroughly investigated in ALS. The aim of this study was to determine whether gene fusions are present in ALS. METHODS: Gene fusions were identified using STAR Fusion v1.10.0 software in bulk RNA-Seq data from human postmortem samples from publicly available data sets from Target ALS and the New York Genome Center ALS Consortium. RESULTS: We report the presence of gene fusion events in several brain regions as well as in spinal cord samples in ALS. Although most gene fusions were intra-chromosomal events between neighboring genes and present in both ALS and control samples, there was a significantly greater number of unique gene fusions in ALS compared to controls. Lastly, we identified specific gene fusions with a significant burden in ALS, that were absent from both control samples and known cancer gene fusion databases. DISCUSSION: Collectively, our findings reveal an enrichment of gene fusions in ALS and suggest that these events may be an additional genetic cause linked to ALS pathogenesis.

Age-Dependent Increase in Tau Phosphorylation at Serine 396 in Huntington's Disease Prefrontal Cortex.

BACKGROUND: To date, it is still controversial whether tau phosphorylation plays a role in Huntington's disease (HD), as previous studies demonstrated either no alterations or increases in phosphorylated tau (pTau) in HD postmortem brain and mouse models. OBJECTIVE: The goal of this study was to determine whether total tau and pTau levels are altered in HD. METHODS: Immunohistochemistry, cellular fractionations, and western blots were used to measure total tau and pTau levels in a large cohort of HD and control postmortem prefrontal cortex (PFC). Furthermore, western blots were performed to assess tau, and pTau levels in HD and control isogenic embryonic stem cell (ESC)-derived cortical neurons and neuronal stem cells (NSCs). Similarly, western blots were used to assess tau and pTau levels in HttQ111 and transgenic R6/2 mice. Lastly, total tau levels were assessed in HD and healthy control plasma using Quanterix Simoa assay. RESULTS: Our results revealed that, while there was no difference in total tau or pTau levels in HD PFC compared to controls, the levels of tau phosphorylated at S396 were increased in PFC samples from HD patients 60 years or older at time of death. Additionally, tau and pTau levels were not changed in HD ESC-derived cortical neurons and NSCs. Similarly, total tau or pTau levels were not altered in HttQ111 and transgenic R6/2 mice compared to wild-type littermates. Lastly, tau levels were not changed in plasma from a small cohort of HD patients compared to controls. CONCLUSIONS: Together these findings demonstrate that pTau-S396 levels increase significantly with age in HD PFC.

Photoactivatable Agonist-Antagonist Pair as a Tool for Precise Spatiotemporal Control of Serotonin Receptor 2C Signaling.

Orthogonal recreation of the signaling profile of a chemical synapse is a current challenge in neuroscience. This is due in part to the kinetics of synaptic signaling, where neurotransmitters are rapidly released and quickly cleared by active reuptake machinery. One strategy to produce a rapid rise in an orthogonally controlled signal is via photocaged compounds. In this work, photocaged compounds are employed to recreate both the rapid rise and equally rapid fall in activation at a chemical synapse. Specifically, a complementary pair of photocages based on BODIPY were conjugated to a 5-HT2C subtype-selective agonist, WAY-161503, and antagonist, N-desmethylclozapine, to generate "caged" versions of these drugs. These conjugates release the bioactive drug upon illumination with green light (agonist) or red light (antagonist). We report on the synthesis, characterization, and bioactivity testing of the conjugates against the 5-HT2C receptor. We then characterize the kinetics of photolysis quantitatively using HPLC and qualitatively in cell culture conditions stimulating live cells. The compounds are shown to be stable in the dark for 48 h at room temperature, yet photolyze rapidly when irradiated with visible light. In live cells expressing the 5-HT2C receptor, precise spatiotemporal control of the degree and length of calcium signaling is demonstrated. By loading both compounds in tandem and leveraging spectral multiplexing as a noninvasive method to control local small-molecule drug availability, we can reproducibly initiate and suppress intracellular calcium flux on a timescale not possible by traditional methods of drug dosing. These tools enable a greater spatiotemporal control of 5-HT2C modulation and will allow for more detailed studies of the receptors' signaling, interactions with other proteins, and native physiology.

Age-dependent increase in tau phosphorylation at serine 396 in Huntington's disease pre-frontal cortex.

Background: To date, it is still controversial whether tau phosphorylation plays a role in Huntington's disease (HD), as previous studies demonstrated either no alterations or increases in phosphorylated tau (pTau) in HD post-mortem brain and mouse models. Objectives: The goal of this study was to determine whether total tau and pTau levels are altered in HD. Methods: Immunohistochemistry, cellular fractionations, and western blots were used to measure tau and pTau levels in a large cohort of HD and control post-mortem prefrontal cortex (PFC). Furthermore, western blots were performed to assess tau, and pTau levels in HD and control isogenic embryonic stem cell (ESC)-derived cortical neurons and neuronal stem cells (NSCs). Similarly, western blots were used to assess tau and pTau in Htt Q111 and transgenic R6/2 mice. Lastly, total tau levels were assessed in HD and healthy control plasma using Quanterix Simoa assay. Results: Our results revealed that, while there was no difference in tau or pTau levels in HD PFC compared to controls, tau phosphorylated at S396 levels were increased in PFC samples from HD patients 60 years or older at time of death. Additionally, tau and pTau levels were not changed in HD ESC-derived cortical neurons and NSCs. Similarly, tau or pTau levels were not altered in Htt Q111 and transgenic R6/2 mice compared to wild-type littermates. Lastly, tau levels were not changed in plasma from a small cohort of HD patients compared to controls. Conclusion: Together these findings demonstrate that pTau-S396 levels increase significantly with age in HD PFC.

Developing Photoaffinity Probes for Dopamine Receptor D2 to Determine Targets of Parkinson's Disease Drugs.

Dopaminergic pathways control highly consequential aspects of physiology and behavior. One of the most therapeutically important and best-studied receptors in these pathways is dopamine receptor D2 (DRD2). Unfortunately, DRD2 is challenging to study with traditional molecular biological techniques, and most drugs designed to target DRD2 are ligands for many other receptors. Here, we developed probes able to both covalently bind to DRD2 using photoaffinity labeling and provide a chemical handle for detection or affinity purification. These probes behaved like good DRD2 agonists in traditional biochemical assays and were able to perform in chemical-biological assays of cell and receptor labeling. Rat whole brain labeling and affinity enrichment using the probes permitted proteomic analysis of the probes' interacting proteins. Bioinformatic study of the hits revealed that the probes bound noncanonically targeted proteins in Parkinson's disease network as well as the retrograde endocannabinoid signaling, neuronal nitric oxide synthase, muscarinic acetylcholine receptor M1, GABA receptor, and dopamine receptor D1 (DRD1) signaling networks. Follow-up analysis may yield insights into how this pathway relates specifically to Parkinson's disease symptoms or provide new targets for treatments. This work reinforces the notion that the combination of chemical biology and omics-based approaches provides a broad picture of a molecule's "interactome" and may also give insight into the pleiotropy of effects observed for a drug or perhaps indicate new applications.

Distinct representation of cue-outcome association by D1 and D2 neurons in the ventral striatum's olfactory tubercle.

Positive and negative associations acquired through olfactory experience are thought to be especially strong and long-lasting. The conserved direct olfactory sensory input to the ventral striatal olfactory tubercle (OT) and its convergence with dense dopaminergic input to the OT could underlie this privileged form of associative memory, but how this process occurs is not well understood. We imaged the activity of the two canonical types of striatal neurons, expressing D1- or D2-type dopamine receptors, in the OT at cellular resolution while mice learned odor-outcome associations ranging from aversive to rewarding. D1 and D2 neurons both responded to rewarding and aversive odors. D1 neurons in the OT robustly and bidirectionally represented odor valence, responding similarly to odors predicting similar outcomes regardless of odor identity. This valence representation persisted even in the absence of a licking response to the odors and in the absence of the outcomes, indicating a true transformation of odor sensory information by D1 OT neurons. In contrast, D2 neuronal representation of the odor-outcome associations was weaker, contingent on a licking response by the mouse, and D2 neurons were more selective for odor identity than valence. Stimulus valence coding in the OT was modality-sensitive, with separate sets of D1 neurons responding to odors and sounds predicting the same outcomes, suggesting that integration of multimodal valence information happens downstream of the OT. Our results point to distinct representation of identity and valence of odor stimuli by D1 and D2 neurons in the OT.

Novel genetic variants in MAPT and alterations in tau phosphorylation in amyotrophic lateral sclerosis post-mortem motor cortex and cerebrospinal fluid.

Although the molecular mechanisms underlying amyotrophic lateral sclerosis (ALS) are not yet fully understood, several studies report alterations in tau phosphorylation in both sporadic and familial ALS. Recently, we have demonstrated that phosphorylated tau at S396 (pTau-S396) is mislocalized to synapses in ALS motor cortex (mCTX) and contributes to mitochondrial dysfunction. Here, we demonstrate that while there was no overall increase in total tau, pTau-S396, and pTau-S404 in ALS post-mortem mCTX, total tau and pTau-S396 were increased in C9ORF72-ALS. Additionally, there was a significant decrease in pTau-T181 in ALS mCTX compared controls. Furthermore, we leveraged the ALS Knowledge Portal and Project MinE data sets and identified ALS-specific genetic variants across MAPT, the gene encoding tau. Lastly, assessment of cerebrospinal fluid (CSF) samples revealed a significant increase in total tau levels in bulbar-onset ALS together with a decrease in CSF pTau-T181:tau ratio in all ALS samples, as reported previously. While increases in CSF tau levels correlated with a faster disease progression as measured by the revised ALS functional rating scale (ALSFRS-R), decreases in CSF pTau-T181:tau ratio correlated with a slower disease progression, suggesting that CSF total tau and pTau-T181 ratio may serve as biomarkers of disease in ALS. Our findings highlight the potential role of pTau-T181 in ALS, as decreases in CSF pTau-T181:tau ratio may reflect the significant decrease in pTau-T181 in post-mortem mCTX. Taken together, these results indicate that tau phosphorylation is altered in ALS post-mortem mCTX as well as in CSF and, importantly, the newly described pathogenic or likely pathogenic variants identified in MAPT in this study are adjacent to T181 and S396 phosphorylation sites further highlighting the potential role of these tau functional domains in ALS.

Targeting Tau Mitigates Mitochondrial Fragmentation and Oxidative Stress in Amyotrophic Lateral Sclerosis.

Understanding the mechanisms underlying amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Previous studies have demonstrated that mitochondrial dysfunction is a key pathogenetic event in ALS. Interestingly, studies in Alzheimer's disease (AD) post-mortem brain and animal models link alterations in mitochondrial function to interactions between hyperphosphorylated tau and dynamin-related protein 1 (DRP1), the GTPase involved in mitochondrial fission. Recent evidence suggest that tau may be involved in ALS pathogenesis, therefore, we sought to determine whether hyperphosphorylated tau may lead to mitochondrial fragmentation and dysfunction in ALS and whether reducing tau may provide a novel therapeutic approach. Our findings demonstrated that pTau-S396 is mis-localized to synapses in post-mortem motor cortex (mCTX) across ALS subtypes. Additionally, the treatment with ALS synaptoneurosomes (SNs), enriched in pTau-S396, increased oxidative stress, induced mitochondrial fragmentation, and altered mitochondrial connectivity without affecting cell survival in vitro. Furthermore, pTau-S396 interacted with DRP1, and similar to pTau-S396, DRP1 accumulated in SNs across ALS subtypes, suggesting increases in mitochondrial fragmentation in ALS. As previously reported, electron microscopy revealed a significant decrease in mitochondria density and length in ALS mCTX. Lastly, reducing tau levels with QC-01-175, a selective tau degrader, prevented ALS SNs-induced mitochondrial fragmentation and oxidative stress in vitro. Collectively, our findings suggest that increases in pTau-S396 may lead to mitochondrial fragmentation and oxidative stress in ALS and decreasing tau may provide a novel strategy to mitigate mitochondrial dysfunction in ALS. pTau-S396 mis-localizes to synapses in ALS. ALS synaptoneurosomes (SNs), enriched in pTau-S396, increase oxidative stress and induce mitochondrial fragmentation in vitro. pTau-S396 interacts with the pro-fission GTPase DRP1 in ALS. Reducing tau with a selective degrader, QC-01-175, mitigates ALS SNs-induced mitochondrial fragmentation and increases in oxidative stress in vitro.

Testosterone Treatment Mimics Seasonal Downregulation of Dopamine Innervation in the Auditory System of Female Midshipman Fish.

In seasonally breeding vertebrates, hormones coordinate changes in nervous system structure and function to facilitate reproductive readiness and success. Steroid hormones often exert their effects indirectly via regulation of neuromodulators, which in turn can coordinate the modulation of sensory input with appropriate motor output. Female plainfin midshipman fish (Porichthys notatus) undergo increased peripheral auditory sensitivity in time for the summer breeding season, improving their ability to detect mates, which is regulated by steroid hormones. Reproductive females also show differences in catecholaminergic innervation of auditory circuitry compared with winter, non-reproductive females as measured by tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholaminergic synthesis. Importantly, catecholaminergic input to the inner ear from a dopaminergic-specific forebrain nucleus is decreased in the summer and dopamine inhibits the sensitivity of the inner ear, suggesting that gonadal steroids may alter auditory sensitivity by regulating dopamine innervation. In this study, we gonadectomized non-reproductive females, implanted them with estradiol (E2) or testosterone (T), and measured TH immunoreactive (TH-ir) fibers in auditory nuclei where catecholaminergic innervation was previously shown to be seasonally plastic. We found that treatment with T, but not E2, reduced TH-ir innervation in the auditory hindbrain. T-treatment also reduced TH-ir fibers in the forebrain dopaminergic cell group that projects to the inner ear, and likely to the auditory hindbrain. Higher T plasma in the treatment group was correlated with reduced-ir TH terminals in the inner ear. These T-treatment induced changes in TH-ir fibers mimic the seasonal downregulation of dopamine in the midshipman inner ear and provide evidence that steroid hormone regulation of peripheral auditory sensitivity is mediated, in part, by dopamine.

Catecholaminergic connectivity to the inner ear, central auditory, and vocal motor circuitry in the plainfin midshipman fish porichthys notatus.

Although the neuroanatomical distribution of catecholaminergic (CA) neurons has been well documented across all vertebrate classes, few studies have examined CA connectivity to physiologically and anatomically identified neural circuitry that controls behavior. The goal of this study was to characterize CA distribution in the brain and inner ear of the plainfin midshipman fish (Porichthys notatus) with particular emphasis on their relationship with anatomically labeled circuitry that both produces and encodes social acoustic signals in this species. Neurobiotin labeling of the main auditory end organ, the saccule, combined with tyrosine hydroxylase immunofluorescence (TH-ir) revealed a strong CA innervation of both the peripheral and central auditory system. Diencephalic TH-ir neurons in the periventricular posterior tuberculum, known to be dopaminergic, send ascending projections to the ventral telencephalon and prominent descending projections to vocal-acoustic integration sites, notably the hindbrain octavolateralis efferent nucleus, as well as onto the base of hair cells in the saccule via nerve VIII. Neurobiotin backfills of the vocal nerve in combination with TH-ir revealed CA terminals on all components of the vocal pattern generator, which appears to largely originate from local TH-ir neurons but may include input from diencephalic projections as well. This study provides strong neuroanatomical evidence that catecholamines are important modulators of both auditory and vocal circuitry and acoustic-driven social behavior in midshipman fish. This demonstration of TH-ir terminals in the main end organ of hearing in a nonmammalian vertebrate suggests a conserved and important anatomical and functional role for dopamine in normal audition.

Synthesis and electrical properties of (Bi(1/2)Na(1/2))TiO3 (BNT) ferroelectric thin films by liquid sprayed mist chemical vapor deposition technique.

This study aims to synthesize lead-free ferroelectric material, (Bi(1/2)Na(1/2))TiO3 using the Liquid Sprayed Mist Chemical Vapor Deposition (LSMCVD) technique. The mist of precursor solution was vaporized and deposited on two different substrates of Si(100) and (111)Pt/TiO2/SiO2/Si(100) in an oxygen atmosphere. The deposition temperature and time were varied in the range of 400-600 degrees C and 30-90 min. (Bi(1/2)Na(1/2))TiO3 thin film had preferred orientations of (110). The thickness of the thin film deposited was 35-162 nm. The remnant polarization (2Pr) and the dielectric constant were 4.6-16.8 microC/cm2, 325-350, respectively.