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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

BV-2 Microglial Cells Overexpressing C9orf72 Hexanucleotide Repeat Expansion Produce DPR Proteins and Show Normal Functionality but No RNA Foci.

Hexanucleotide repeat expansion (HRE) in the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common genetic cause underpinning frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). It leads to the accumulation of toxic RNA foci and various dipeptide repeat (DPR) proteins into cells. These C9orf72 HRE-specific hallmarks are abundant in neurons. So far, the role of microglia, the immune cells of the brain, in C9orf72 HRE-associated FTLD/ALS is unclear. In this study, we overexpressed C9orf72 HRE of a pathological length in the BV-2 microglial cell line and used biochemical methods and fluorescence imaging to investigate its effects on their phenotype, viability, and functionality. We found that BV-2 cells expressing the C9orf72 HRE presented strong expression of specific DPR proteins but no sense RNA foci. Transiently increased levels of cytoplasmic TAR DNA-binding protein 43 (TDP-43), slightly altered levels of p62 and lysosome-associated membrane protein (LAMP) 2A, and reduced levels of polyubiquitinylated proteins, but no signs of cell death were detected in HRE overexpressing cells. Overexpression of the C9orf72 HRE did not affect BV-2 cell phagocytic activity or response to an inflammatory stimulus, nor did it shift their RNA profile toward disease-associated microglia. These findings suggest that DPR proteins do not affect microglial cell viability or functionality in BV-2 cells. However, additional studies in other models are required to further elucidate the role of C9orf72 HRE in microglia.

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.

C9orf72 Proteins Regulate Autophagy and Undergo Autophagosomal or Proteasomal Degradation in a Cell Type-Dependent Manner.

Dysfunctional autophagy or ubiquitin-proteasome system (UPS) are suggested to underlie abnormal protein aggregation in neurodegenerative diseases. Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS)-associated C9orf72 is implicated in autophagy, but whether it activates or inhibits autophagy is partially controversial. Here, we utilized knockdown or overexpression of C9orf72 in mouse N2a neuroblastoma cells or cultured neurons to elucidate the potential role of C9orf72 proteins in autophagy and UPS. Induction of autophagy in C9orf72 knockdown N2a cells led to decreased LC3BI to LC3BII conversion, p62 degradation, and formation of LC3-containing autophagosomes, suggesting compromised autophagy. Proteasomal activity was slightly decreased. No changes in autophagy nor proteasomal activity in C9orf72-overexpressing N2a cells were observed. However, in these cells, autophagy induction by serum starvation or rapamycin led to significantly decreased C9orf72 levels. The decreased levels of C9orf72 in serum-starved N2a cells were restored by the proteasomal inhibitor lactacystin, but not by the autophagy inhibitor bafilomycin A1 (BafA1) treatment. These data suggest that C9orf72 undergoes proteasomal degradation in N2a cells during autophagy. Lactacystin significantly elevated C9orf72 levels in N2a cells and neurons, further suggesting UPS-mediated regulation. In rapamycin and BafA1-treated neurons, C9orf72 levels were significantly increased. Altogether, these findings corroborate the previously suggested regulatory role for C9orf72 in autophagy and suggest cell type-dependent regulation of C9orf72 levels via UPS and/or autophagy.

PSEN1ΔE9, APPswe, and APOE4 Confer Disparate Phenotypes in Human iPSC-Derived Microglia.

Here we elucidate the effect of Alzheimer disease (AD)-predisposing genetic backgrounds, APOE4, PSEN1ΔE9, and APPswe, on functionality of human microglia-like cells (iMGLs). We present a physiologically relevant high-yield protocol for producing iMGLs from induced pluripotent stem cells. Differentiation is directed with small molecules through primitive erythromyeloid progenitors to re-create microglial ontogeny from yolk sac. The iMGLs express microglial signature genes and respond to ADP with intracellular Ca2+ release distinguishing them from macrophages. Using 16 iPSC lines from healthy donors, AD patients and isogenic controls, we reveal that the APOE4 genotype has a profound impact on several aspects of microglial functionality, whereas PSEN1ΔE9 and APPswe mutations trigger minor alterations. The APOE4 genotype impairs phagocytosis, migration, and metabolic activity of iMGLs but exacerbates their cytokine secretion. This indicates that APOE4 iMGLs are fundamentally unable to mount normal microglial functionality in AD.

Astrocytes and Microglia as Potential Contributors to the Pathogenesis of C9orf72 Repeat Expansion-Associated FTLD and ALS.

Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases with a complex, but often overlapping, genetic and pathobiological background and thus they are considered to form a disease spectrum. Although neurons are the principal cells affected in FTLD and ALS, increasing amount of evidence has recently proposed that other central nervous system-resident cells, including microglia and astrocytes, may also play roles in neurodegeneration in these diseases. Therefore, deciphering the mechanisms underlying the disease pathogenesis in different types of brain cells is fundamental in order to understand the etiology of these disorders. The major genetic cause of FTLD and ALS is a hexanucleotide repeat expansion (HRE) in the intronic region of the C9orf72 gene. In neurons, specific pathological hallmarks, including decreased expression of the C9orf72 RNA and proteins and generation of toxic RNA and protein species, and their downstream effects have been linked to C9orf72 HRE-associated FTLD and ALS. In contrast, it is still poorly known to which extent these pathological changes are presented in other brain cells. Here, we summarize the current literature on the potential role of astrocytes and microglia in C9orf72 HRE-linked FTLD and ALS and discuss their possible phenotypic alterations and neurotoxic mechanisms that may contribute to neurodegeneration in these diseases.

Interrelationship between the Levels of C9orf72 and Amyloid-ß Protein Precursor and Amyloid-ß in Human Cells and Brain Samples.

A subset of C9orf72 repeat expansion-carrying frontotemporal dementia patients display an Alzheimer-like decrease in cerebrospinal fluid amyloid-ß (Aß) biomarker levels. We report that downregulation of C9orf72 in non-neuronal human cells overexpressing amyloid-ß protein precursor (AßPP) resulted in increased levels of secreted AßPP fragments and Aß, while levels of AßPP or its C-terminal fragments (CTFs) remained unchanged. In neuronal cells, AßPP and C83 CTF levels were decreased upon C9orf72 knockdown, but those of secreted AßPP fragments or Aß remained unchanged. C9orf72 protein levels significantly increased in human brain with advancing neurofibrillary pathology and positively correlated with brain Aß42 levels. Our data suggest that altered C9orf72 levels may lead to cell-type specific alterations in AßPP processing, but warrant further studies to clarify the underlying mechanisms.