TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS.

Cytoplasmic accumulation of TDP-43 is a disease hallmark for many cases of amyotrophic lateral sclerosis (ALS), associated with a neuroinflammatory cytokine profile related to upregulation of nuclear factor κB (NF-κB) and type I interferon (IFN) pathways. Here we show that this inflammation is driven by the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) when TDP-43 invades mitochondria and releases DNA via the permeability transition pore. Pharmacologic inhibition or genetic deletion of cGAS and its downstream signaling partner STING prevents upregulation of NF-κB and type I IFN induced by TDP-43 in induced pluripotent stem cell (iPSC)-derived motor neurons and in TDP-43 mutant mice. Finally, we document elevated levels of the specific cGAS signaling metabolite cGAMP in spinal cord samples from patients, which may be a biomarker of mtDNA release and cGAS/STING activation in ALS. Our results identify mtDNA release and cGAS/STING activation as critical determinants of TDP-43-associated pathology and demonstrate the potential for targeting this pathway in ALS.

Obesity Drives STAT-1-Dependent NASH and STAT-3-Dependent HCC.

Obesity is a major driver of cancer, especially hepatocellular carcinoma (HCC). The prevailing view is that non-alcoholic steatohepatitis (NASH) and fibrosis or cirrhosis are required for HCC in obesity. Here, we report that NASH and fibrosis and HCC in obesity can be dissociated. We show that the oxidative hepatic environment in obesity inactivates the STAT-1 and STAT-3 phosphatase T cell protein tyrosine phosphatase (TCPTP) and increases STAT-1 and STAT-3 signaling. TCPTP deletion in hepatocytes promoted T cell recruitment and ensuing NASH and fibrosis as well as HCC in obese C57BL/6 mice that normally do not develop NASH and fibrosis or HCC. Attenuating the enhanced STAT-1 signaling prevented T cell recruitment and NASH and fibrosis but did not prevent HCC. By contrast, correcting STAT-3 signaling prevented HCC without affecting NASH and fibrosis. TCPTP-deletion in hepatocytes also markedly accelerated HCC in mice treated with a chemical carcinogen that promotes HCC without NASH and fibrosis. Our studies reveal how obesity-associated hepatic oxidative stress can independently contribute to the pathogenesis of NASH, fibrosis, and HCC.

Control of Glucocorticoid Receptor Levels by PTEN Establishes a Failsafe Mechanism for Tumor Suppression.

The PTEN tumor suppressor controls cell death and survival by regulating functions of various molecular targets. While the role of PTEN lipid-phosphatase activity on PtdIns(3,4,5)P3 and inhibition of PI3K pathway is well characterized, the biological relevance of PTEN protein-phosphatase activity remains undefined. Here, using knockin (KI) mice harboring cancer-associated and functionally relevant missense mutations, we show that although loss of PTEN lipid-phosphatase function cooperates with oncogenic PI3K to promote rapid mammary tumorigenesis, the additional loss of PTEN protein-phosphatase activity triggered an extensive cell death response evident in early and advanced mammary tumors. Omics and drug-targeting studies revealed that PI3Ks act to reduce glucocorticoid receptor (GR) levels, which are rescued by loss of PTEN protein-phosphatase activity to restrain cell survival. Thus, we find that the dual regulation of GR by PI3K and PTEN functions as a rheostat that can be exploited for the treatment of PTEN loss-driven cancers.

Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer's Disease Across Distinct Brain Regions.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex etiology influenced by confounding factors such as genetic polymorphisms, age, sex, and race. Traditionally, AD research has not prioritized these influences, resulting in dramatically skewed cohorts such as three times the number of Apolipoprotein E (APOE) ε4-allele carriers in AD relative to healthy cohorts. Thus, the resulting molecular changes in AD have previously been complicated by the influence of apolipoprotein E disparities. To explore how apolipoprotein E polymorphism influences AD progression, 62 post-mortem patients consisting of 33 AD and 29 controls (Ctrl) were studied to balance the number of ε4-allele carriers and facilitate a molecular comparison of the apolipoprotein E genotype. Lipid and protein perturbations were assessed across AD diagnosed brains compared to Ctrl brains, ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s and APOE4- for non-ε4 carriers), and differences in ε3ε3 and ε3ε4 Ctrl brains across two brain regions (frontal cortex (FCX) and cerebellum (CBM)). The region-specific influences of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction and cell proteostasis at the core of AD pathophysiology in the post-mortem brains, indicating these two processes may be influenced by genotypic differences and brain morphology.

Chronic traumatic encephalopathy neuropathologic change in former Australian rugby players.

AIMS: We applied the 2021 consensus criteria for both chronic traumatic encephalopathy neuropathological change and traumatic encephalopathy syndrome in a small case series of six former elite-level Australian rugby code players. METHODS: Neuropathological assessment of these cases was carried out at the Sydney and Victorian Brain Banks. Clinical data were collected via clinical interviews and health questionnaires completed by the participants and/or their next of kin, and neuropsychological testing was conducted with participants who were capable of completing this testing. RESULTS: All cases exhibited progressive cognitive impairment during life. Chronic traumatic encephalopathy neuropathological change was identified in four out of the six cases. However, coexisting neuropathologies were common, with limbic-predominant age-related TDP-43 encephalopathy and ageing-related tau astrogliopathy seen in all cases, intermediate or high Alzheimer's disease neuropathological change seen in four cases and hippocampal sclerosis seen in two of the six cases. CONCLUSION: The presence of multiple neuropathologies in these cases complicates clinical diagnostic efforts for traumatic encephalopathy syndrome. It will be important for further clinicopathological studies on larger groups to report all neuropathological comorbidities found in cases diagnosed with either chronic traumatic encephalopathy neuropathological change and/or traumatic encephalopathy syndrome.

Cardiovascular Magnetic Resonance for Rejection Surveillance After Cardiac Transplantation.

BACKGROUND: Endomyocardial biopsy (EMB) is the gold standard method for surveillance of acute cardiac allograft rejection (ACAR) despite its invasive nature. Cardiovascular magnetic resonance (CMR)-based myocardial tissue characterization allows detection of myocarditis. The feasibility of CMR-based surveillance for ACAR-induced myocarditis in the first year after heart transplantation is currently undescribed. METHODS: CMR-based multiparametric mapping was initially assessed in a prospective cross-sectional fashion to establish agreement between CMR- and EMB-based ACAR and to determine CMR cutoff values between rejection grades. A prospective randomized noninferiority pilot study was then undertaken in adult orthotopic heart transplant recipients who were randomized at 4 weeks after orthotopic heart transplantation to either CMR- or EMB-based rejection surveillance. Clinical end points were assessed at 52 weeks. RESULTS: Four hundred one CMR studies and 354 EMB procedures were performed in 106 participants. Forty heart transplant recipients were randomized. CMR-based multiparametric assessment was highly reproducible and reliable at detecting ACAR (area under the curve, 0.92; sensitivity, 93%; specificity, 92%; negative predictive value, 99%) with greater specificity and negative predictive value than either T1 or T2 parametric CMR mapping alone. High-grade rejection occurred in similar numbers of patients in each randomized group (CMR, n=7; EMB, n=8; P=0.74). Despite similarities in immunosuppression requirements, kidney function, and mortality between groups, the rates of hospitalization (9 of 20 [45%] versus 18 of 20 [90%]; odds ratio, 0.091; P=0.006) and infection (7 of 20 [35%] versus 14 of 20 [70%]; odds ratio, 0.192; P=0,019) were lower in the CMR group. On 15 occasions (6%), patients who were randomized to the CMR arm underwent EMB for clarification or logistic reasons, representing a 94% reduction in the requirement for EMB-based surveillance. CONCLUSIONS: A noninvasive CMR-based surveillance strategy for ACAR in the first year after orthotopic heart transplantation is feasible compared with EMB-based surveillance. REGISTRATION: HREC/13/SVH/66 and HREC/17/SVH/80. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY: ACTRN12618000672257.

11C-PiB PET can underestimate brain amyloid-ß burden when cotton wool plaques are numerous.

Individuals with familial Alzheimer's disease due to PSEN1 mutations develop high cortical fibrillar amyloid-ß load but often have lower cortical 11C-Pittsburgh compound B (PiB) retention than Individuals with sporadic Alzheimer's disease. We hypothesized this is influenced by limited interactions of Pittsburgh compound B with cotton wool plaques, an amyloid-ß plaque type common in familial Alzheimer's disease but rare in sporadic Alzheimer's disease. Histological sections of frontal and temporal cortex, caudate nucleus and cerebellum were obtained from 14 cases with sporadic Alzheimer's disease, 12 cases with familial Alzheimer's disease due to PSEN1 mutations, two relatives of a PSEN1 mutation carrier but without genotype information and three non-Alzheimer's disease cases. Sections were processed immunohistochemically using amyloid-ß-targeting antibodies and the fluorescent amyloid stains cyano-PiB and X-34. Plaque load was quantified by percentage area analysis. Frozen homogenates from the same brain regions from five sporadic Alzheimer's disease and three familial Alzheimer's disease cases were analysed for 3H-PiB in vitro binding and concentrations of amyloid-ß1-40 and amyloid-ß1-42. Nine sporadic Alzheimer's disease, three familial Alzheimer's disease and three non-Alzheimer's disease participants had 11C-PiB PET with standardized uptake value ratios calculated using the cerebellum as the reference region. Cotton wool plaques were present in the neocortex of all familial Alzheimer's disease cases and one sporadic Alzheimer's disease case, in the caudate nucleus from four familial Alzheimer's disease cases, but not in the cerebellum. Cotton wool plaques immunolabelled robustly with 4G8 and amyloid-ß42 antibodies but weakly with amyloid-ß40 and amyloid-ßN3pE antibodies and had only background cyano-PiB fluorescence despite labelling with X-34. Relative to amyloid-ß plaque load, cyano-Pittsburgh compound B plaque load was similar in sporadic Alzheimer's disease while in familial Alzheimer's disease it was lower in the neocortex and the caudate nucleus. In both regions, insoluble amyloid-ß1-42 and amyloid-ß1-40 concentrations were similar in familial Alzheimer's disease and sporadic Alzheimer's disease groups, while 3H-PiB binding was lower in the familial Alzheimer's disease than the sporadic Alzheimer's disease group. Higher amyloid-ß1-42 concentration associated with higher 3H-PiB binding in sporadic Alzheimer's disease but not familial Alzheimer's disease. 11C-PiB retention correlated with region-matched post-mortem amyloid-ß plaque load; however, familial Alzheimer's disease cases with abundant cotton wool plaques had lower 11C-PiB retention than sporadic Alzheimer's disease cases with similar amyloid-ß plaque loads. PiB has limited ability to detect amyloid-ß aggregates in cotton wool plaques and may underestimate total amyloid-ß plaque burden in brain regions with abundant cotton wool plaques.

PtdIns(3,4,5)P3-dependent Rac exchanger 1 (P-Rex1) promotes mammary tumor initiation and metastasis.

The Rac-GEF, P-Rex1, activates Rac1 signaling downstream of G protein-coupled receptors and PI3K. Increased P-Rex1 expression promotes melanoma progression; however, its role in breast cancer is complex, with differing reports of the effect of its expression on disease outcome. To address this we analyzed human databases, undertook gene array expression analysis, and generated unique murine models of P-Rex1 gain or loss of function. Analysis of PREX1 mRNA expression in breast cancer cDNA arrays and a METABRIC cohort revealed that higher PREX1 mRNA in ER+ve/luminal tumors was associated with poor outcome in luminal B cancers. Prex1 deletion in MMTV-neu or MMTV-PyMT mice reduced Rac1 activation in vivo and improved survival. High level MMTV-driven transgenic PREX1 expression resulted in apicobasal polarity defects and increased mammary epithelial cell proliferation associated with hyperplasia and development of de novo mammary tumors. MMTV-PREX1 expression in MMTV-neu mice increased tumor initiation and enhanced metastasis in vivo, but had no effect on primary tumor growth. Pharmacological inhibition of Rac1 or MEK1/2 reduced P-Rex1-driven tumoroid formation and cell invasion. Therefore, P-Rex1 can act as an oncogene and cooperate with HER2/neu to enhance breast cancer initiation and metastasis, despite having no effect on primary tumor growth.

Severe NAD(P)HX Dehydratase (NAXD) Neurometabolic Syndrome May Present in Adulthood after Mild Head Trauma.

We have previously reported that pathogenic variants in a key metabolite repair enzyme NAXD cause a lethal neurodegenerative condition triggered by episodes of fever in young children. However, the clinical and genetic spectrum of NAXD deficiency is broadening as our understanding of the disease expands and as more cases are identified. Here, we report the oldest known individual succumbing to NAXD-related neurometabolic crisis, at 32 years of age. The clinical deterioration and demise of this individual were likely triggered by mild head trauma. This patient had a novel homozygous NAXD variant [NM_001242882.1:c.441+3A>G:p.?] that induces the mis-splicing of the majority of NAXD transcripts, leaving only trace levels of canonically spliced NAXD mRNA, and protein levels below the detection threshold by proteomic analysis. Accumulation of damaged NADH, the substrate of NAXD, could be detected in the fibroblasts of the patient. In agreement with prior anecdotal reports in paediatric patients, niacin-based treatment also partly alleviated some clinical symptoms in this adult patient. The present study extends our understanding of NAXD deficiency by uncovering shared mitochondrial proteomic signatures between the adult and our previously reported paediatric NAXD cases, with reduced levels of respiratory complexes I and IV as well as the mitoribosome, and the upregulation of mitochondrial apoptotic pathways. Importantly, we highlight that head trauma in adults, in addition to paediatric fever or illness, may precipitate neurometabolic crises associated with pathogenic NAXD variants.

Does Data-Independent Acquisition Data Contain Hidden Gems? A Case Study Related to Alzheimer's Disease.

One of the potential benefits of using data-independent acquisition (DIA) proteomics protocols is that information not originally targeted by the study may be present and discovered by subsequent analysis. Herein, we reanalyzed DIA data originally recorded for global proteomic analysis to look for isomerized peptides, which occur as a result of spontaneous chemical modifications to long-lived proteins. Examination of a large set of human brain samples revealed a striking relationship between Alzheimer's disease (AD) status and isomerization of aspartic acid in a peptide from tau. Relative to controls, a surprising increase in isomer abundance was found in both autosomal dominant and sporadic AD samples. To explore potential mechanisms that might account for these observations, quantitative analysis of proteins related to isomerization repair and autophagy was performed. Differences consistent with reduced autophagic flux in AD-related samples relative to controls were found for numerous proteins, including most notably p62, a recognized indicator of autophagic inhibition. These results suggest, but do not conclusively demonstrate, that lower autophagic flux may be strongly associated with loss of function in AD brains. This study illustrates that DIA data may contain unforeseen results of interest and may be particularly useful for pilot studies investigating new research directions. In this case, a promising target for future investigations into the therapy and prevention of AD has been identified.

Sex-dependent effects of amyloid precursor-like protein 2 in the SOD1-G37R transgenic mouse model of MND.

Motor neurone disease (MND) is a neurodegenerative disorder characterised by progressive destruction of motor neurons, muscle paralysis and death. The amyloid precursor protein (APP) is highly expressed in the central nervous system and has been shown to modulate disease outcomes in MND. APP is part of a gene family that includes the amyloid precursor-like protein 1 (APLP1) and 2 (APLP2) genes. In the present study, we investigated the role of APLP2 in MND through the examination of human spinal cord tissue and by crossing APLP2 knockout mice with the superoxide dismutase 1 (SOD1-G37R) transgenic mouse model of MND. We found the expression of APLP2 is elevated in the spinal cord from human cases of MND and that this feature of the human disease is reproduced in SOD1-G37R mice at the End-stage of their MND-like phenotype progression. APLP2 deletion in SOD1-G37R mice significantly delayed disease progression and increased the survival of female SOD1-G37R mice. Molecular and biochemical analysis showed female SOD1-G37R:APLP2-/- mice displayed improved innervation of the neuromuscular junction, ameliorated atrophy of muscle fibres with increased APP protein expression levels in the gastrocnemius muscle. These results indicate a sex-dependent role for APLP2 in mutant SOD1-mediated MND and further support the APP family as a potential target for further investigation into the cause and regulation of MND.

Biallelic hypomorphic variants in ALDH1A2 cause a novel lethal human multiple congenital anomaly syndrome encompassing diaphragmatic, pulmonary, and cardiovascular defects.

This study shows a causal association between ALDH1A2 variants and a novel, severe multiple congenital anomaly syndrome in humans that is neonatally lethal due to associated pulmonary hypoplasia and respiratory failure. In two families, exome sequencing identified compound heterozygous missense variants in ALDH1A2. ALDH1A2 is involved in the conversion of retinol (vitamin A) into retinoic acid (RA), which is an essential regulator of diaphragm and cardiovascular formation during embryogenesis. Reduced RA causes cardiovascular, diaphragmatic, and associated pulmonary defects in several animal models, matching the phenotype observed in our patients. In silico protein modeling showed probable impairment of ALDH1A2 for three of the four substitutions. In vitro studies show a reduction of RA. Few pathogenic variants in genes encoding components of the retinoic signaling pathway have been described to date, likely due to embryonic lethality. Thus, this study contributes significantly to knowledge of the role of this pathway in human diaphragm and cardiovascular development and disease. Some clinical features in our patients are also observed in Fryns syndrome (MIM# 229850), syndromic microphthalmia 9 (MIM# 601186), and DiGeorge syndrome (MIM# 188400). Patients with similar clinical features who are genetically undiagnosed should be tested for recessive ALDH1A2-deficient malformation syndrome.

Evaluation of FGFR targeting in breast cancer through interrogation of patient-derived models.

BACKGROUND: Particular breast cancer subtypes pose a clinical challenge due to limited targeted therapeutic options and/or poor responses to the existing targeted therapies. While cell lines provide useful pre-clinical models, patient-derived xenografts (PDX) and organoids (PDO) provide significant advantages, including maintenance of genetic and phenotypic heterogeneity, 3D architecture and for PDX, tumor-stroma interactions. In this study, we applied an integrated multi-omic approach across panels of breast cancer PDXs and PDOs in order to identify candidate therapeutic targets, with a major focus on specific FGFRs. METHODS: MS-based phosphoproteomics, RNAseq, WES and Western blotting were used to characterize aberrantly activated protein kinases and effects of specific FGFR inhibitors. PDX and PDO were treated with the selective tyrosine kinase inhibitors AZD4547 (FGFR1-3) and BLU9931 (FGFR4). FGFR4 expression in cancer tissue samples and PDOs was assessed by immunohistochemistry. METABRIC and TCGA datasets were interrogated to identify specific FGFR alterations and their association with breast cancer subtype and patient survival. RESULTS: Phosphoproteomic profiling across 18 triple-negative breast cancers (TNBC) and 1 luminal B PDX revealed considerable heterogeneity in kinase activation, but 1/3 of PDX exhibited enhanced phosphorylation of FGFR1, FGFR2 or FGFR4. One TNBC PDX with high FGFR2 activation was exquisitely sensitive to AZD4547. Integrated 'omic analysis revealed a novel FGFR2-SKI fusion that comprised the majority of FGFR2 joined to the C-terminal region of SKI containing the coiled-coil domains. High FGFR4 phosphorylation characterized a luminal B PDX model and treatment with BLU9931 significantly decreased tumor growth. Phosphoproteomic and transcriptomic analyses confirmed on-target action of the two anti-FGFR drugs and also revealed novel effects on the spliceosome, metabolism and extracellular matrix (AZD4547) and RIG-I-like and NOD-like receptor signaling (BLU9931). Interrogation of public datasets revealed FGFR2 amplification, fusion or mutation in TNBC and other breast cancer subtypes, while FGFR4 overexpression and amplification occurred in all breast cancer subtypes and were associated with poor prognosis. Characterization of a PDO panel identified a luminal A PDO with high FGFR4 expression that was sensitive to BLU9931 treatment, further highlighting FGFR4 as a potential therapeutic target. CONCLUSIONS: This work highlights how patient-derived models of human breast cancer provide powerful platforms for therapeutic target identification and analysis of drug action, and also the potential of specific FGFRs, including FGFR4, as targets for precision treatment.

Quantification of metallothionein-III in brain tissues using liquid chromatography tandem mass spectrometry.

Metallothioneins (MTs) are crucial for metal ion homeostasis in mammalian cells. Specialized mass spectrometry methods have been developed to detect MTs in tissue extracts, though facile methods with scalable throughput are lacking. To improve analytical throughput and repeatability, we developed a standardised liquid chromatography tandem mass spectrometry (LC-MS/MS) method for robust determination of metallothionein-3 (MT3) that is amenable to microplate processing. This method uses standard protein digestion conditions with commercially available reagents and commonly practiced reversed-phase chromatography, detecting MT3 at low ng/mL levels in human brain tissue extracts. We found that trypsin digestion largely underestimated MT3 levels, whereas endopeptidase Lys-C yielded vastly higher signals with low replicate variance. The choice of target peptide was critical for accurate MT3 detection - a peptide in the α-domain yielded the most robust signals. We demonstrate the utility of this method by comparing the expression of MT3 in post-mortem brain tissues of a cohort of Alzheimer's disease (AD) individuals and age-matched controls.

The Selective Expansion and Targeted Accumulation of Bone Marrow-Derived Macrophages Drive Cardiac Vasculitis.

The adult heart contains macrophages derived from both embryonic and adult bone marrow (BM)-derived precursors. This population diversity prompted us to explore how distinct macrophage subsets localize within the heart, and their relative contributions in cardiac disease. In this study, using the reciprocal expression of Lyve-1 and Ccr2 to distinguish macrophages with distinct origins, we show that, in the steady state, both embryonic (Lyvepos) and BM-derived (Ccr2pos) macrophages populate the major vessels of the heart in mice and humans. However, cardiac macrophage populations are markedly perturbed by inflammation. In a mouse model of Kawasaki disease, BM-derived macrophages preferentially increase during acute cardiac inflammation and selectively accumulate around major cardiac vessels. The accumulation of BM-derived macrophages coincides with the loss of their embryonic counterparts and is an initiating, essential step in the emergence of subsequent cardiac vasculitis in this experimental model. Finally, we demonstrate that the accumulation of Ccr2pos macrophages (and the development of vasculitis) occurs in close proximity to a population of Ccr2 chemokine ligand-producing epicardial cells, suggesting that the epicardium may be involved in localizing inflammation to cardiac vessels. Collectively, our findings identify the perivascular accumulation of BM-derived macrophages as pivotal in the pathogenesis of cardiac vasculitis and provide evidence about the mechanisms governing their recruitment to the heart.

Clinical and Neuropathological Features Associated With Loss of RAB39B.

BACKGROUND: Pathogenic variants in the small GTPase Ras Analogue in Brain 39b (RAB39B) have been linked to the development of early-onset parkinsonism. The study was aimed at delineating the clinical and neuropathological features associated with a previously reported pathogenic variant in RAB39B (c.503C>A p.T168K) and testing for dysregulation of RAB39B in idiopathic PD. METHODS: Clinical details of a male individual hemizygous for the T168K variant were collected by systematic review of medical records. Neuropathological studies of fixed brain tissue were performed and steady-state RAB39B levels were determined by western blot analysis. RESULTS: Neuropathological examination showed extensive dopaminergic neuron loss, widespread Lewy pathology, and iron accumulation in the substantia nigra. Additional pathology was observed in the hippocampus and thalamus. Western blot analysis demonstrated that the T168K variant results in loss of RAB39B. In individuals with idiopathic PD (n = 10, 6 male/4 female), steady-state RAB39B was significantly reduced in the prefrontal cortex and substantia nigra. CONCLUSIONS: T168K RAB39B is unstable in vivo and associated with dopaminergic neuron loss and Lewy pathology. Dysregulation of RAB39B in the prefrontal cortex and substantia nigra of individuals with idiopathic PD potentially implicates the protein more broadly in the pathological mechanisms underlying PD and related Lewy body disorders. © 2020 International Parkinson and Movement Disorder Society.

Copper Bis(thiosemicarbazonato)-stilbenyl Complexes That Bind to Amyloid-ß Plaques.

Alzheimer's disease is characterized by the presence of extracellular amyloid-ß plaques. Positron emission tomography (PET) imaging with tracers radiolabeled with positron-emitting radionuclides that bind to amyloid-ß plaques can assist in the diagnosis of Alzheimer's disease. With the goal of designing new imaging agents radiolabeled with positron-emitting copper-64 radionuclides that bind to amyloid-ß plaques, a family of bis(thiosemicarbazone) ligands with appended substituted stilbenyl functional groups has been prepared. The ligands form charge-neutral and stable complexes with copper(II). The new ligands can be radiolabeled with copper-64 at room temperature. Two lead complexes were demonstrated to bind to amyloid-ß plaques present in post-mortem brain tissue from subjects with clinically diagnosed Alzheimer's disease and crossed the blood-brain barrier in mice. The work presented here provides strategies to prepare compounds with radionuclides of copper that can be used for targeted brain PET imaging.

C9orf72 intermediate repeats are associated with corticobasal degeneration, increased C9orf72 expression and disruption of autophagy.

Microsatellite repeat expansion disease loci can exhibit pleiotropic clinical and biological effects depending on repeat length. Large expansions in C9orf72 (100s-1000s of units) are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). However, whether intermediate expansions also contribute to neurodegenerative disease is not well understood. Several studies have identified intermediate repeats in Parkinson's disease patients, but the association was not found in autopsy-confirmed cases. We hypothesized that intermediate C9orf72 repeats are a genetic risk factor for corticobasal degeneration (CBD), a neurodegenerative disease that can be clinically similar to Parkinson's but has distinct tau protein pathology. Indeed, intermediate C9orf72 repeats were significantly enriched in autopsy-proven CBD (n = 354 cases, odds ratio = 3.59, p = 0.00024). While large C9orf72 repeat expansions are known to decrease C9orf72 expression, intermediate C9orf72 repeats result in increased C9orf72 expression in human brain tissue and CRISPR/cas9 knockin iPSC-derived neural progenitor cells. In contrast to cases of FTD/ALS with large C9orf72 expansions, CBD with intermediate C9orf72 repeats was not associated with pathologic RNA foci or dipeptide repeat protein aggregates. Knock-in cells with intermediate repeats exhibit numerous changes in gene expression pathways relating to vesicle trafficking and autophagy. Additionally, overexpression of C9orf72 without the repeat expansion leads to defects in autophagy under nutrient starvation conditions. These results raise the possibility that therapeutic strategies to reduce C9orf72 expression may be beneficial for the treatment of CBD.

Potential Diagnostic Imaging of Alzheimer's Disease with Copper-64 Complexes That Bind to Amyloid-ß Plaques.

Amyloid-ß plaques, consisting of aggregated amyloid-ß peptides, are one of the pathological hallmarks of Alzheimer's disease. Copper complexes formed using positron-emitting copper radionuclides that cross the blood-brain barrier and bind to specific molecular targets offer the possibility of noninvasive diagnostic imaging using positron emission tomography. New thiosemicarbazone-pyridylhydrazone based ligands that incorporate pyridyl-benzofuran functional groups designed to bind amyloid-ß plaques have been synthesized. The ligands form stable complexes with copper(II) ( Kd = 10-18 M) and can be radiolabeled with copper-64 at room temperature. Subtle changes to the periphery of the ligand backbone alter the metabolic stability of the complexes in mouse and human liver microsomes, and influenced the ability of the complexes to cross the blood-brain barrier in mice. A lead complex was selected based on possessing the best metabolic stability and brain uptake in mice. Synthesis of this lead complex with isotopically enriched copper-65 allowed us to show that the complex bound to amyloid-ß plaques present in post-mortem human brain tissue using laser ablation-inductively coupled plasma-mass spectrometry. This work provides insight into strategies to target metal complexes to amyloid-ß plaques, and how small modifications to ligands can dramatically alter the metabolic stability of metal complexes as well as their ability to cross the blood-brain barrier.

Anatomic location of primary melanoma: Survival differences and sun exposure.

BACKGROUND: Anatomic location of melanoma has been shown to independently influence melanoma-specific survival (MSS). OBJECTIVE: We aimed to compare the MSS of specific anatomic subsites and between chronically, intermittently, and rarely sun-exposed sites. METHODS: A prospective cohort study was performed of primary invasive cutaneous melanomas with known thickness and location reviewed at a tertiary referral center over 21 years. RESULTS: Overall, 3570 primary cutaneous invasive melanoma cases were included. After adjustment for clinicopathologic variables (including thickness, ulceration, mitotic rate, sex, age, and subtype), posterior scalp melanoma was associated with worse MSS (hazard ratio [HR], 2.46; 95% confidence interval [CI], 1.38-4.40) compared with the upper back, whereas melanoma on the thighs, forearms/hands, and anterior upper arms had better MSS. Intermittent (HR, 0.56; 95% CI, 0.41-0.76) and chronically sun-exposed sites (HR, 0.70; 95% CI, 0.51-0.96) had improved survival compared with rarely exposed sites on multivariate analysis. LIMITATIONS: Potential selection bias of a tertiary referral center selecting for advanced cases. CONCLUSION: Altered MSS in the posterior scalp, thighs, forearms, hands, and anterior upper arms appears to be independent of clinicopathologic factors. Results were similar for both sexes and age groups. The posterior scalp should be considered a poor prognosis site.