Threshold of heteroplasmic truncating MT-ATP6 mutation in reprogramming, Notch hyperactivation and motor neuron metabolism.

Mutations in mitochondrial DNA encoded subunit of ATP synthase, MT-ATP6, are frequent causes of neurological mitochondrial diseases with a range of phenotypes from Leigh syndrome and NARP to ataxias and neuropathies. Here we investigated the functional consequences of an unusual heteroplasmic truncating mutation m.9154C>T in MT-ATP6, which caused peripheral neuropathy, ataxia and IgA nephropathy. ATP synthase not only generates cellular ATP, but its dimerization is required for mitochondrial cristae formation. Accordingly, the MT-ATP6 truncating mutation impaired the assembly of ATP synthase and disrupted cristae morphology, supporting our molecular dynamics simulations that predicted destabilized a/c subunit subcomplex. Next, we modeled the effects of the truncating mutation using patient-specific induced pluripotent stem cells. Unexpectedly, depending on mutation heteroplasmy level, the truncation showed multiple threshold effects in cellular reprogramming, neurogenesis and in metabolism of mature motor neurons (MN). Interestingly, MN differentiation beyond progenitor stage was impaired by Notch hyperactivation in the MT-ATP6 mutant, but not by rotenone-induced inhibition of mitochondrial respiration, suggesting that altered mitochondrial morphology contributed to Notch hyperactivation. Finally, we also identified a lower mutation threshold for a metabolic shift in mature MN, affecting lactate utilization, which may be relevant for understanding the mechanisms of mitochondrial involvement in peripheral motor neuropathies. These results establish a critical and disease-relevant role for ATP synthase in human cell fate decisions and neuronal metabolism.

Rare PMP22 variants in mild to severe neuropathy uncorrelated to plasma GDF15 or neurofilament light.

Charcot-Marie-Tooth disease (CMT) is a heterogeneous set of hereditary neuropathies whose genetic causes are not fully understood. Here, we characterize three previously unknown variants in PMP22 and assess their effect on the recently described potential CMT biomarkers' growth differentiation factor 15 (GDF15) and neurofilament light (NFL): first, a heterozygous PMP22 c.178G > A (p.Glu60Lys) in one mother-son pair with adult-onset mild axonal neuropathy. The variant led to abnormal splicing, confirmed in fibroblasts by reverse transcription PCR. Second, a de novo PMP22 c.35A > C (p.His12Pro), and third, a heterozygous 3.2 kb deletion predicting loss of exon 4. The latter two had severe CMT and ultrasonography showing strong nerve enlargement similar to a previous case of exon 4 loss due to a larger deletion. We further studied patients with PMP22 duplication (CMT1A) finding slightly elevated plasma NFL, as measured by the single molecule array immunoassay (SIMOA). In addition, plasma GDF15, as measured by ELISA, correlated with symptom severity for CMT1A. However, in the severely affected individuals with PMP22 exon 4 deletion or p.His12Pro, these biomarkers were within the range of variability of CMT1A and controls, although they had more pronounced nerve hypertrophy. This study adds p.His12Pro and confirms PMP22 exon 4 deletion as causes of severe CMT, whereas the previously unknown splice variant p.Glu60Lys leads to mild axonal neuropathy. Our results suggest that GDF15 and NFL do not distinguish CMT1A from advanced hypertrophic neuropathy caused by rare PMP22 variants.

Deficient neurotransmitter systems and synaptic function in frontotemporal lobar degeneration-Insights into disease mechanisms and current therapeutic approaches.

Frontotemporal lobar degeneration (FTLD) comprises a heterogenous group of fatal neurodegenerative diseases and, to date, no validated diagnostic or prognostic biomarkers or effective disease-modifying therapies exist for the different clinical or genetic subtypes of FTLD. Current treatment strategies rely on the off-label use of medications for symptomatic treatment. Changes in several neurotransmitter systems including the glutamatergic, GABAergic, dopaminergic, and serotonergic systems have been reported in FTLD spectrum disease patients. Many FTLD-related clinical and neuropsychiatric symptoms such as aggressive and compulsive behaviour, agitation, as well as altered eating habits and hyperorality can be explained by disturbances in these neurotransmitter systems, suggesting that their targeting might possibly offer new therapeutic options for treating patients with FTLD. This review summarizes the present knowledge on neurotransmitter system deficits and synaptic dysfunction in model systems and patients harbouring the most common genetic causes of FTLD, the hexanucleotide repeat expansion in C9orf72 and mutations in the granulin (GRN) and microtubule-associated protein tau (MAPT) genes. We also describe the current pharmacological treatment options for FLTD that target different neurotransmitter systems.

C9orf72 hexanucleotide repeat expansion leads to altered neuronal and dendritic spine morphology and synaptic dysfunction.

Frontotemporal lobar degeneration (FTLD) comprises a heterogenous group of progressive neurodegenerative syndromes. To date, no validated biomarkers or effective disease-modifying therapies exist for the different clinical or genetic subtypes of FTLD. The most common genetic cause underlying FTLD and amyotrophic lateral sclerosis (ALS) is a hexanucleotide repeat expansion in the C9orf72 gene (C9-HRE). FTLD is accompanied by changes in several neurotransmitter systems, including the glutamatergic, GABAergic, dopaminergic, and serotonergic systems and many clinical symptoms can be explained by disturbances in these systems. Here, we aimed to elucidate the effects of the C9-HRE on synaptic function, molecular composition of synapses, and dendritic spine morphology. We overexpressed the pathological C9-HRE in cultured E18 mouse primary hippocampal neurons and characterized the pathological, morphological, and functional changes by biochemical methods, confocal microscopy, and live cell calcium imaging. The C9-HRE-expressing neurons were confirmed to display the pathological RNA foci and DPR proteins. C9-HRE expression led to significant changes in dendritic spine morphologies, as indicated by decreased number of mushroom-type spines and increased number of stubby and thin spines, as well as diminished neuronal branching. These morphological changes were accompanied by concomitantly enhanced susceptibility of the neurons to glutamate-induced excitotoxicity as well as augmented and prolonged responses to excitatory stimuli by glutamate and depolarizing potassium chloride as compared to control neurons. Mechanistically, the hyperexcitation phenotype in the C9-HRE-expressing neurons was found to be underlain by increased activity of extrasynaptic GluN2B-containing N-methyl-d-aspartate (NMDA) receptors. Our results are in accordance with the idea suggesting that C9-HRE is associated with enhanced excitotoxicity and synaptic dysfunction. Thus, therapeutic interventions targeted to alleviate synaptic disturbances might offer efficient avenues for the treatment of patients with C9-HRE-associated FTLD.

GFAP as a biomarker in frontotemporal dementia and primary psychiatric disorders: diagnostic and prognostic performance.

BACKGROUND: Frontotemporal lobar degeneration (FTLD) and primary psychiatric disorders (PPD) are characterised by overlapping clinical features but different aetiologies. Here, we assessed for the first time the potential of blood glial fibrillar acidic protein (GFAP), marker of astrogliosis, as a discriminative and prognostic tool in FTLD and PPD. METHODS: The levels of GFAP in serum (sGFAP) of patients with FTLD (N=107) and PPD (N=44) and GFAP in whole blood samples (bGFAP) from FTLD (N=10), PPD (N=10) and healthy controls (N=18) were measured. We evaluated whether the sGFAP levels associate with C9orf72 repeat expansion, survival of FTLD and PPD patients, and brain atrophy assessed cross-sectionally and longitudinally by structural T1W MRI. We also examined the correlation between sGFAP and bGFAP levels in a subset of patients. RESULTS: sGFAP and bGFAP levels were elevated in the FTLD group compared with the PPD or control groups. Receiver operating characteristic analysis indicated an excellent diagnostic performance between FTLD and PPD (the area under the curve (AUC)=0.820, 95% CI 0.745 to 0.896). sGFAP and bGFAP levels showed a strong correlation and elevated sGFAP levels significantly associated with atrophy rate in the temporal cortex and predicted shorter survival time in patients with FTLD. No association with C9orf72 repeat expansion was detected. CONCLUSIONS: sGFAP enabled differentiation of patients with FTLD and PPD and associated with shorter survival and more severe brain atrophy rate in patients with FTLD. These results suggest that blood-based GFAP represents a minimally invasive and useful biomarker in the differential diagnostics between patients with FTLD and PPD and in evaluating disease progression and astrogliosis in FTLD.

DHCR24 exerts neuroprotection upon inflammation-induced neuronal death.

BACKGROUND: DHCR24, involved in the de novo synthesis of cholesterol and protection of neuronal cells against different stress conditions, has been shown to be selectively downregulated in neurons of the affected brain areas in Alzheimer's disease. METHODS: Here, we investigated whether the overexpression of DHCR24 protects neurons against inflammation-induced neuronal death using co-cultures of mouse embryonic primary cortical neurons and BV2 microglial cells upon acute neuroinflammation. Moreover, the effects of DHCR24 overexpression on dendritic spine density and morphology in cultured mature mouse hippocampal neurons and on the outcome measures of ischemia-induced brain damage in vivo in mice were assessed. RESULTS: Overexpression of DHCR24 reduced the loss of neurons under inflammation elicited by LPS and IFN-γ treatment in co-cultures of mouse neurons and BV2 microglial cells but did not affect the production of neuroinflammatory mediators, total cellular cholesterol levels, or the activity of proteins linked with neuroprotective signaling. Conversely, the levels of post-synaptic cell adhesion protein neuroligin-1 were significantly increased upon the overexpression of DHCR24 in basal growth conditions. Augmentation of DHCR24 also increased the total number of dendritic spines and the proportion of mushroom spines in mature mouse hippocampal neurons. In vivo, overexpression of DHCR24 in striatum reduced the lesion size measured by MRI in a mouse model of transient focal ischemia. CONCLUSIONS: These results suggest that the augmentation of DHCR24 levels provides neuroprotection in acute stress conditions, which lead to neuronal loss in vitro and in vivo.

Obesity-induced hyperleptinemia improves survival and immune response in a murine model of sepsis.

BACKGROUND: Obesity is a growing health problem and associated with immune dysfunction. Sepsis is defined as systemic inflammatory response syndrome that occurs during infection. Excessive inflammation combined with immune dysfunction can lead to multiorgan damage and death. METHODS: The authors investigated the influence of a class 1 obesity (body mass index between 30 and 34.9) on immune function and outcome in sepsis and the role of leptin on the immune response. The authors used a long-term high-fat-diet feeding model (12 weeks) on C57Bl/6 mice (n = 100) and controls on standard diet (n = 140) followed by a polymicrobial sepsis induced by cecal ligation and puncture. RESULTS: The authors show that class 1 obesity is connected to significant higher serum leptin levels (data are mean ± SEM) (5.7 ± 1.2 vs. 2.7 ± 0.2 ng/ml; n = 5; P = 0.033) and improved innate immune response followed by significant better survival rate in sepsis (71.4%, n = 10 vs. 10%, n = 14; P < 0.0001). Additional sepsis-induced increases in leptin levels stabilize body temperature and are associated with a controlled immune response in a time-dependent and protective manner. Furthermore, leptin treatment of normal-weight septic mice with relative hypoleptinemia (n = 35) also significantly stabilizes body temperature, improves cellular immune response, and reduces proinflammatory cytokine response resulting in improved survival (30%; n = 10). CONCLUSIONS: Relative hyperleptinemia of class 1 obesity or induced by treatment is protective in sepsis. Leptin seems to play a regulatory role in the immune system in sepsis, and treatment of relative hypoleptinemia could offer a new way of an individual sepsis therapy.

Organotypic Hippocampal Slice Cultures from Adult Tauopathy Mice and Theragnostic Evaluation of Nanomaterial Phospho-TAU Antibody-Conjugates.

Organotypic slice culture models surpass conventional in vitro methods in many aspects. They retain all tissue-resident cell types and tissue hierarchy. For studying multifactorial neurodegenerative diseases such as tauopathies, it is crucial to maintain cellular crosstalk in an accessible model system. Organotypic slice cultures from postnatal tissue are an established research tool, but adult tissue-originating systems are missing, yet necessary, as young tissue-originating systems cannot fully model adult or senescent brains. To establish an adult-originating slice culture system for tauopathy studies, we made hippocampal slice cultures from transgenic 5-month-old hTau.P301S mice. In addition to the comprehensive characterization, we set out to test a novel antibody for hyperphosphorylated TAU (pTAU, B6), with and without a nanomaterial conjugate. Adult hippocampal slices retained intact hippocampal layers, astrocytes, and functional microglia during culturing. The P301S-slice neurons expressed pTAU throughout the granular cell layer and secreted pTAU to the culture medium, whereas the wildtype slices did not. Additionally, cytotoxicity and inflammation-related determinants were increased in the P301S slices. Using fluorescence microscopy, we showed target engagement of the B6 antibody to pTAU-expressing neurons and a subtle but consistent decrease in intracellular pTAU with the B6 treatment. Collectively, this tauopathy slice culture model enables measuring the extracellular and intracellular effects of different mechanistic or therapeutic manipulations on TAU pathology in adult tissue without the hindrance of the blood-brain barrier.

Serum Cathepsin S Levels Do Not Show Alterations in Different Clinical, Neuropathological, or Genetic Subtypes of Frontotemporal Dementia Patients nor in Comparison to Healthy Control Individuals.

Frontotemporal dementia (FTD) can manifest as diverse clinical phenotypes and is frequently caused by mutations in different genes, complicating differential diagnosis. This underlines the urgent need for valid biomarkers. Altered lysosomal and immune functions proposedly contribute to FTD pathogenesis. Cathepsins, including cathepsin S, are enzymes preferentially expressed in brain in microglia, which influence lysosomal and immune function. Here, we examined whether alterations in serum cathepsin S levels associate with specific clinical, genetic, or neuropathological FTD subgroups, but no such alterations were observed. However, further research on other lysosomal proteins may reveal new biologically relevant biomarkers in FTD.

Cortico-spinal tDCS in amyotrophic lateral sclerosis: A randomized, double-blind, sham-controlled trial followed by an open-label phase.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive disease for which no curative treatment is currently available. OBJECTIVE: This study aimed to investigate whether cortico-spinal transcranial direct current stimulation (tDCS) could mitigate symptoms in ALS patients via a randomized, double-blind, sham-controlled trial, followed by an open-label phase. METHODS: Thirty-one participants were randomized into two groups for the initial controlled phase. At baseline (T0), Group 1 received placebo stimulation (sham tDCS), while Group 2 received cortico-spinal stimulation (real tDCS) for five days/week for two weeks (T1), with an 8-week (T2) follow-up (randomized, double-blind, sham-controlled phase). At the 24-week follow-up (T3), all participants (Groups 1 and 2) received a second treatment of anodal bilateral motor cortex and cathodal spinal stimulation (real tDCS) for five days/week for two weeks (T4). Follow-up evaluations were performed at 32-weeks (T5) and 48-weeks (T6) (open-label phase). At each time point, clinical assessment, blood sampling, and intracortical connectivity measures using transcranial magnetic stimulation (TMS) were evaluated. Additionally, we evaluated survival rates. RESULTS: Compared to sham stimulation, cortico-spinal tDCS significantly improved global strength, caregiver burden, and quality of life scores, which correlated with the restoration of intracortical connectivity measures. Serum neurofilament light levels decreased among patients who underwent real tDCS but not in those receiving sham tDCS. The number of completed 2-week tDCS treatments significantly influenced patient survival. CONCLUSIONS: Cortico-spinal tDCS may represent a promising therapeutic and rehabilitative approach for patients with ALS. Further larger-scale studies are necessary to evaluate whether tDCS could potentially impact patient survival. CLINICAL TRIAL REGISTRATION: NCT04293484.

Changed adipocytokine concentrations in colorectal tumor patients and morbidly obese patients compared to healthy controls.

BACKGROUND: Obesity has been associated with increased incidence of colorectal cancer. Adipose tissue dysfunction accompanied with alterations in the release of adipocytokines has been proposed to contribute to cancer pathogenesis and progression. The aim of this study was to analyze plasma concentrations of several adipose tissue expressed hormones in colorectal cancer patients (CRC) and morbidly obese (MO) patients and to compare these concentrations to clinicopathological parameters. METHODS: Plasma concentrations of adiponectin, resistin, leptin, active plasminogen activator inhibitor (PAI)-1, monocyte chemotactic protein (MCP)-1, interleukin (IL)-1 alpha, and tumor necrosis factor (TNF)-alpha were determined in 67 patients operated on for CRC (31 rectal cancers, 36 colon cancers), 37 patients operated on for morbid obesity and 60 healthy blood donors (BD). RESULTS: Compared to BD, leptin concentrations were lowered in CRC patients whereas those of MO patients were elevated. Adiponectin concentrations were only lowered in MO patients. Concentrations of MCP-1, PAI-1, and IL-1 alpha were elevated in both CRC and MO patients, while resistin and TNF-alpha were similarly expressed in MO and CRC patients compared to BD. Resistin concentrations positively correlated with tumor staging (p<0.002) and grading (p=0.015) of rectal tumor patients. CONCLUSIONS: The results suggest that both MO and CRC have low-grade inflammation as part of their etiology.

c-Jun N-terminal kinase 2 suppresses pancreatic cancer growth and invasion and is opposed by c-Jun N-terminal kinase 1.

The c-Jun N-terminal protein kinases (JNKs) JNK1 and JNK2 can act as either tumor suppressors or pro-oncogenic kinases in human cancers. The isoform-specific roles for JNK1 and JNK2 in human pancreatic cancer are still unclear, the question which should be addressed in this project. Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 clones were established either expressing either JNK1 or -2 shRNA in a stable manner. Basal anchorage-dependent and -independent cell growth, single-cell movement, and invasion using the Boyden chamber assay were analyzed. Xenograft growth was assessed using an orthotopic mouse model. All seven tested pancreatic cancer cell lines expressed JNKs as did human pancreatic cancer samples determined by immunohistochemistry. Pharmacological, unspecific JNK inhibition (SP600125) reduced cell growth of all cell lines but PANC-1. Especially inhibition of JNK2 resulted in overall increased oncogenic potential with increased proliferation and invasion, associated with alterations in cytoskeleton structure. Specific inhibition of JNK1 revealed opposing functions. Overall, JNK1 and JNK2 can exert different functions in human pancreatic cancer and act as counter players for tumor invasion. Specifically modulating the activity of JNKs may be of potential therapeutic interest in the future.

Serum total TDP-43 levels are decreased in frontotemporal dementia patients with C9orf72 repeat expansion or concomitant motoneuron disease phenotype.

BACKGROUND: Frontotemporal dementia (FTD) covers a spectrum of neurodegenerative disorders with various clinical and neuropathological subtypes. The two major pathological proteins accumulating in the brains of FTD patients, depending on their genetic background, are TDP-43 and tau. We aimed to evaluate whether total TDP-43 levels measured from the serum associate with the genotype or clinical phenotype of the FTD patients and whether serum TDP-43 provides prognostic or diagnostic value in the FTD spectrum disorders. METHODS: The study cohort included 254 participants with a clinical diagnosis of FTD (including all major genotypes and clinical phenotypes) and 105 cognitively healthy controls. Serum total TDP-43 levels measured with a single-molecule array (Simoa) were compared within the FTD group according to the genotype, clinical phenotype, and predicted neuropathological subtype of the patients. We also evaluated the associations between the TDP-43 levels and disease severity or survival in FTD. RESULTS: Total TDP-43 levels in the serum were significantly lower in the FTD group as compared to the healthy control group (275.3 pg/mL vs. 361.8 pg/mL, B = 0.181, 95%CI = 0.014-0.348, p = 0.034). The lowest TDP-43 levels were observed in the subgroup of FTD patients harboring predicted TDP-43 brain pathology (FTD-TDP, 241.4 pg/mL). The low levels in the FTD-TDP group were especially driven by C9orf72 repeat expansion carriers (169.2 pg/mL) and FTD patients with concomitant motoneuron disease (FTD-MND, 113.3 pg/mL), whereas GRN mutation carriers did not show decreased TDP-43 levels (328.6 pg/mL). Serum TDP-43 levels showed no correlation with disease severity nor progression in FTD. CONCLUSIONS: Our results indicate that the total levels of TDP-43 in the serum are decreased especially in FTD patients with the C9orf72 repeat expansion or FTD-MND phenotype, both subtypes strongly associated with TDP-43 type B brain pathology. Serum-based measurement of TDP-43 could represent a useful tool in indicating C9orf72 repeat expansion and FTD-MND-related TDP-43 neuropathology for future diagnostics and intervention studies.

CNS leptin action modulates immune response and survival in sepsis.

Sepsis describes a complex clinical syndrome that results from an infection, setting off a cascade of systemic inflammatory responses that can lead to multiple organ failure and death. Leptin is a 16 kDa adipokine that, among its multiple known effects, is involved in regulating immune function. Here we demonstrate that leptin deficiency in ob/ob mice leads to higher mortality and more severe organ damage in a standard model of sepsis in mice [cecal ligation and puncture (CLP)]. Moreover, systemic leptin replacement improved the immune response to CLP. Based on the molecular mechanisms of leptin regulation of energy metabolism and reproductive function, we hypothesized that leptin acts in the CNS to efficiently coordinate peripheral immune defense in sepsis. We now report that leptin signaling in the brain increases survival during sepsis in leptin-deficient as well as in wild-type mice and that endogenous CNS leptin action is required for an adequate systemic immune response. These findings reveal the existence of a relevant neuroendocrine control of systemic immune defense and suggest a possible therapeutic potential for leptin analogs in infectious disease.

Expression of C9orf72 hexanucleotide repeat expansion leads to formation of RNA foci and dipeptide repeat proteins but does not influence autophagy or proteasomal function in neuronal cells.

C9orf72 hexanucleotide repeat expansion (HRE) is the major genetic cause underpinning frontotemporal lobar degeneration (FLTD) and amyotrophic lateral sclerosis (ALS). C9orf72 HRE-associated pathogenesis involves both loss-of-function, through reduced C9orf72 levels, and gain-of-function mechanisms, including formation of RNA foci and generation of dipeptide repeat (DPR) proteins. In addition, dysfunctional protein degradation pathways, i.e. autophagy and ubiquitin-proteasome system (UPS), are suggested. Our aim was to study the gain-of-function mechanisms in the context of the function of protein degradation pathways as well as the regulation of the DPR proteins through these pathways. To this end, we expressed the pathological HRE in neuronal N2a cells and mouse primary cortical neurons. Protein degradation pathways were modulated to induce or block autophagy or to inhibit UPS. In addition, proteasomal activity was assessed. The C9orf72 HRE-expressing N2a cells and neurons were confirmed to produce RNA foci and DPR proteins, predominantly the Poly-GP proteins. However, the presence of these pathological hallmarks did not result in alterations in autophagy or proteasomal activity in either of the studied cell types. In N2a cells, Poly-GP proteins appeared in soluble forms and Lactacystin-mediated UPS inhibition increased their levels, indicating proteasomal regulation. Similar effects were not observed in cortical neurons, where the Poly-GP proteins formed also higher molecular weight forms. These results suggest a cell type-specific morphology and regulation of the DPR proteins. Further studies in other model systems may shed additional light onto the effects of the C9orf72 HRE on cellular protein degradation pathways and the regulation of the DPR protein levels.

Serum GFAP and NfL levels in benign relapsing-remitting multiple sclerosis.

OBJECTIVE: We aimed to investigate serum glial fibrillary acidic protein (GFAP) and serum neurofilament light chain (NfL) levels as potential discriminative biomarkers between benign relapsing-remitting multiple sclerosis (BRRMS) and aggressive relapsing-remitting MS (ARRMS). METHODS: Serum GFAP and NfL levels were analyzed in patients with BRRMS (n = 34), ARRMS (n = 29), and healthy controls (n = 14) by using Single Molecule Array (Simoa). Patients with ARRMS had been treated with highly effective disease-modifying treatments (DMT) (fingolimod or natalizumab). RESULTS: Serum GFAP levels in both BRRMS (median 210.19 pg/ml, IQR 163.69-287.19) and in ARRMS (median 188.60 pg/ml, IQR39.23-244.93) were significantly higher (p = 0.035 and p = 0.034, respectively) compared to healthy controls (median 117.93 pg/ml, IQR 60.28-183.83). Serum GFAP levels did not differ between BRRMS and ARRMS. There were no statistical differences in NfL levels between BRRMS, ARRMS and healthy controls. GFAP level was significantly higher (p = 0.04) in BRRMS without DMT (median 216.04 pg/ml, IQR 188.60-274.79) than in those BRRMS patients who had used DMT (median 196.26 pg/ml, IQR 133.33-325.54). CONCLUSIONS: We found elevated levels of serum GFAP in both BRRMS and ARRMS compared to healthy controls, reflecting astrocytic activation. Serum NfL did not differ between BRRMS and ARRMS, probably due to the stable inflammatory phase of the disease and effective DMT use in ARRMS. Single serum NfL and GFAP measurements cannot separate a patient with BRRMS from effectively treated ARRMS after a long history of the disease, thus consecutive samples are needed in the follow-up.

Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons.

Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot-Marie-Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.

FTLD Patient-Derived Fibroblasts Show Defective Mitochondrial Function and Accumulation of p62.

Frontotemporal lobar degeneration (FTLD) is a clinically, genetically, and neuropathologically heterogeneous group of neurodegenerative syndromes, leading to progressive cognitive dysfunction and frontal and temporal atrophy. C9orf72 hexanucleotide repeat expansion (C9-HRE) is the most common genetic cause of FTLD, but pathogenic mechanisms underlying FTLD are not fully understood. Here, we compared cellular features and functional properties, especially related to protein degradation pathways and mitochondrial function, of FTLD patient-derived skin fibroblasts from C9-HRE carriers and non-carriers and healthy donors. Fibroblasts from C9-HRE carriers were found to produce RNA foci, but no dipeptide repeat proteins, and they showed unchanged levels of C9orf72 mRNA transcripts. The main protein degradation pathways, the ubiquitin-proteasome system and autophagy, did not show alterations between the fibroblasts from C9-HRE-carrying and non-carrying FTLD patients and compared to healthy controls. An increase in the number and size of p62-positive puncta was evident in fibroblasts from both C9-HRE carriers and non-carriers. In addition, several parameters of mitochondrial function, namely, basal and maximal respiration and respiration linked to ATP production, were significantly reduced in the FTLD patient-derived fibroblasts from both C9-HRE carriers and non-carriers. Our findings suggest that FTLD patient-derived fibroblasts, regardless of whether they carry the C9-HRE expansion, show unchanged proteasomal and autophagic function, but significantly impaired mitochondrial function and increased accumulation of p62 when compared to control fibroblasts. These findings suggest the possibility of utilizing FTLD patient-derived fibroblasts as a platform for biomarker discovery and testing of drugs targeted to specific cellular functions, such as mitochondrial respiration.

Age-related decrease in proteasome expression contributes to defective nuclear factor-kappaB activation during hepatic ischemia/reperfusion.

Hepatic ischemia/reperfusion (I/R) leads to liver injury and dysfunction through the initiation of a biphasic inflammatory response that is regulated by the transcription factor nuclear factor kappaB (NF-kappaB). We have previously shown that there is an age-dependent difference in the injury response to hepatic I/R in mice that correlates with divergent activation of NF-kappaB such that young mice have greater NF-kappaB activation, but less injury than old mice. In this study, we investigated the mechanism by which age alters the activation of NF-kappaB in the liver during I/R. Young (4-5 weeks) and old (12-14 months) mice underwent partial hepatic I/R. Livers were obtained for RNA microarray analysis and protein expression assays. Using microarray analysis, we identified age-dependent differences in the expression of genes related to protein ubiquitinylation and the proteasome. In old mice, genes that are involved in the ubiquitin-proteasome pathway were significantly down-regulated during I/R. Consistent with these findings, expression of a critical proteasome subunit, non-adenosine triphosphatase 4 (PSMD4), was reduced in old mice. Expression of the NF-kappaB inhibitory protein, IkappaB alpha, was increased in old mice and was greatly phosphorylated and ubiquitinylated. The data provide strong evidence that the age-related defect in hepatic NF-kappaB signaling during I/R is a result of decreased expression of PSMD4, a proteasome subunit responsible for recognition and recruitment of ubiquitinylated substrates to the proteasome. It appears that decreased PSMD4 expression prevents recruitment of phosphorylated and ubiquitinylated IkappaB alpha to the proteasome, resulting in a defect in NF-kappaB activation.

Frontotemporal dementia: a conference report from the 2nd FinFTD Symposium.

The 2nd FinFTD Symposium was held on 13 September 2019, in Kuopio, Finland, and attracted 80 attendees from six different countries. The program, spanning from molecular mechanisms to biomarkers, prevention, diagnosis and treatment of frontotemporal lobar degeneration and related diseases, provided a great opportunity for researchers, clinicians, healthcare professionals and other participants to discuss about the current status and future directions of frontotemporal lobar degeneration research.