Abstract Body

The accumulation of abnormal forms of the tau protein is a characteristic feature of AD, FTD-tau and numerous other neurodegenerative diseases and syndromes. The initial events that cause tau to dissociate from microtubules and relocate from the axon to the soma are poorly understood. It is also not clear how post translational modifications may play a role in these events, and at what stage, and where, tau first forms aggregates. Using a series of physiologically relevant FTD model systems (mutant MAPT KI mice, and novel human neurons) we have started to explore the events that lead to overt neuronal dysfunction and degeneration both in vivo and in vitro.

Abstract Body

Accumulation of the RNA-binding protein TDP43 into cytoplasmic inclusions are the hallmark of the heterogeneous group of neurodegenerative conditions, frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS-TDP). Based on morphologies, FTLD-TDP pathology can be subclassified into 3 major subtypes with relatively specific clinical and genetic correlations. The molecular mechanisms that lead to and/or trigger TDP43 aggregation and the molecular basis behind the phenotypic heterogeneity remains to be established. Aggregated TDP43 is heavily modified by posttranslational modifications; thus, the main aim of our study is to dissect the role of phosphorylation as regulator of TDP43 functions and in aggregation. To gain insights into TDP43 phosphorylation in distinct functional states, we performed LC-MS/MS analysis of HEK239 cells expressing TDP43 mutants with either impaired RNA binding ability, impaired nuclear import, or upon arsenite treatment to induce stress granules, pathways thought to be involved in pathogenesis. Basic characterization of phosphomimicking or dephosphomimicking TDP43 mutants against more than 30 identified phosphorylation sites revealed alterations in the subcellular distribution and solubility for several sites in the RRM1, RRM2 and prion-like domain. To enable investigation of these phosphorylation events under endogenous expression levels and in human tissues we started an antibody generation program. Novel phosphospecific monoclonal antibodies are established against serin residues 317, 369, 373, 375, 395; antibodies against additional sites are currently characterized. Notably, immunoreactivity profiles by immunohistochemistry for antibodies pS369, 373 and 375 differed between FTLD-TDP subtypes, with labelling of type B+C but not of type A inclusions, suggesting biochemical differences of TDP43 species. Further insights of posttranslational modifications are thought to be crucial to understand disease pathogenesis and are expected to have impact on future development of biomarker assays and disease modifying therapies.

Aims

TDP-43 pathology is prevalent in dementia, but the cell type-specific effects of TDP-43 alterations are not clear. Recent studies suggest that TDP-43 dysfunction in glial cells, including astrocytes, may contribute to disease. However, the effects of astrocytic TDP-43 on neurocognitive processes, astrocytic-neuronal interactions, and neuroimmune responses are not known. Here we investigated the effects of astrocytic TDP-43 on behavior, neural activities, and immune defenses in the brain.

Methods

We analyzed astrocytic TDP-43 distribution in postmortem human brain samples from patients with Alzheimer’s disease (AD) or frontotemporal dementia (FTD). We also used transgenic mice with inducible mutant TDP-43 targeted to astrocytes and complementary AAV-based manipulations in vivo. In mouse models, we performed behavioral characterization across ages, molecular analyses in multiple brain regions, electrophysiological assays, and measurements of neural susceptibility to viral infections. In addition, we used isolated astrocytes, neurons, and astrocyte-neuronal co-cultures to dissect molecular mechanisms and cell-cell interactions.

Results

We found that cases with AD or FTD had diffuse extranuclear TDP-43 accumulation in hippocampal astrocytes. In mouse models, widespread or hippocampus-targeted accumulation in astrocytic TDP-43 caused memory loss and changes in antiviral pathways. These changes were cell-autonomous and included altered antiviral defenses and release of interferon-inducible chemokines. Neurons had elevated levels of the chemokine receptor CXCR3 selectively in excitatory presynaptic terminals. Chronic CXCR3 stimulation caused neuronal hyperexcitability, akin to the effects of astrocytic TDP-43 accumulation in co-cultures and hippocampal slices derived from transgenic mice. Blockade of CXCR3 reduced these aberrant activities and genetic ablation of CXCR3 prevented TDP-43-linked memory loss, implicating aberrant chemokine signaling in cognitive decline.

Conclusions

Astrocytic TDP-43 dysregulation alters antiviral responses and promotes cognitive decline by inducing neuronal hyperexcitability through chemokine-mediated changes in presynaptic function, revealing novel therapeutic targets.

Aims

TDP-43 pathology characterizes most amyotrophic lateral sclerosis (ALS) cases, and ~50% of patients with frontotemporal lobar degeneration (FTLD). In disease, TDP-43, a nuclear protein involved in RNA homeostasis, becomes insoluble and either aggregates in the nucleus or mislocalizes to the cytoplasm, leading to a loss of its nuclear function. Interestingly, TDP-43 proteinopathy can be found in aging brains (termed limbic-predominant, age-related TDP-43 encephalopathy or LATE), and may be concomitant with Alzheimer’s disease (AD) neuropathological changes (LATE/AD) in up to 70% of AD patients. Further, the presence of TDP-43 pathology in AD associates with greater disease severity. Thus, it is imperative that we understand the contributions of TDP-43 to AD.

Methods

We assessed amygdala, hippocampus, and frontal cortex from a cohort of 192 brains. We evaluated the extent of TDP-43 deposition across affected brain regions by using an immunoassay to quantify the levels of insoluble phosphorylated TDP-43 (pTDP-43). TDP-43 dysfunction was evaluated by interrogating the accumulation of TDP-43 regulated cryptic RNAs that have been reported to accumulate in FTLD-TDP.

Results

pTDP-43 similarly accumulated in amygdala and hippocampus of LATE/AD and FTLD-TDP cases, while frontal cortex pTDP-43 accumulation was only observed in FTLD-TDP. Misspliced cryptic TDP-43-regulated RNAs known to accumulate in FTLD-TDP frontal cortex, significantly accumulated in the amygdala and hippocampus of FTLD-TDP cases, as well as in LATE/AD cases, and efficiently discriminated from cases without TDP-43 pathology. The topographic distribution of cryptic RNA accumulation mimicked that of pTDP-43, regardless of TDP-43 subtype classification.

Conclusions

Cryptic RNAs may represent an intriguing new therapeutic and diagnostic target in AD. Further, TDP-43 dysfunction and related changes in cryptic splicing could represent a common molecular mechanism shared between LATE/AD and FTLD-TDP, and potentially other TDP-43 proteinopathies.

Aims

To investigate the safety, tolerability, and efficacy of PR006 (AAV9-GRN) in FTD patients with pathogenic heterogenous GRN mutation (FTD-GRN).

Methods

PR006 (AAV9-GRN) is an investigational gene therapy that delivers a healthy copy of the GRN gene to neurons and other cells in the CNS. In an ongoing open-label Phase I/II trial (NTCT04408625) in subjects with FTD-GRN, PR006 was administered as one time injection to the cisterna magna. Subjects are enrolled for a 1-year main study period with a 4-year follow up period. Safety parameters, biomarkers, clinical scales, and imaging are obtained at baseline and during follow up.

Results

13 subjects received PR006 at 2 dose levels (Table 1). A planned interim analysis was performed after subjects in the low dose cohort finished the 1-year main study period. PR006 administration was generally safe and well-tolerated. The treatment restored cerebral spinal fluid (CSF) progranulin to normal physiological levels (Figure 1) and increased CSF and urinary levels of bis(monoacylglycero)phosphate (BMP), a biomarker indicative of improved lysosomal function, (Figure 2).

Table 1. Baseline Characteristics of Treated Subjects

Conclusions

Interim biomarker analysis of the low- and mid-dose cohorts of the PROCLAIM trial provides evidence suggesting that PR006 may be safe and efficacious in patients with FTD-GRN, thus supporting further clinical development in this patient population.

Aims

Progranulin (PGRN) haploinsufficiency is a major risk factor for frontotemporal lobar degeneration with TDP-43 pathology (FTLD-GRN). Multiple therapeutic strategies are in clinical development to restore PGRN levels in the CNS, including gene therapy. However, major limitations of current gene therapy approaches are their inefficient brain penetrance, biodistribution, and side effects. For these reasons, we aimed to develop an adeno-associated virus (AAV) targeting the liver (L) to achieve sustained peripheral expression of a transferrin receptor (TfR) binding, brain-penetrant (b) PGRN variant (AAV(L):bPGRN).

Methods

We administered a single i.v. AAV(L):bPGRN dose to two established mouse models of FTLD-GRN – Grn knockout and Grn x Tmem106b double knockout (DKO) mice – and analyzed motor performance, lipidomics and transcriptomics, autophagic defects, microglial activation, TDP-43 pathology, and neurodegeneration. We also generated GRN and TMEM106B single- and DKO human induced pluripotent stem cells (hiPSC). Using differentiated microglia and neuron mono- and co-cultures, we established and characterized an in vitro model system recapitulating FTLD-GRN-like pathology.

Results

AAV(L):bPGRN treatment rescued several FTLD-GRN-associated phenotypes, including severe motor deficits, TDP-43 aggregation and phosphorylation, dysfunctional protein degradation, aberrant lipid metabolism, gliosis, and neurodegeneration. Translatability of our findings was confirmed in our novel human in vitro model using co-cultured DKO hiPSC-derived microglia and wild-type hiPSC-derived neurons. As in mice, TDP-43 phosphorylation and hyper-processing, lysosomal dysfunction, and neuronal loss were reduced after treatment with exogenous TfR-binding protein transport vehicle fused to PGRN (PTV:PGRN).

Conclusions

Together, our results show that peripherally administered brain-penetrant PGRN replacement strategies can ameliorate FTLD-GRN-related phenotypes in preclinical models. Our data provide proof of concept for the use of this AAV platform in the treatment of FTLD-GRN and potentially other CNS disorders.

Aims

Amyotrophic lateral sclerosis (ALS) and Frontotemporal Dementia (FTD) are characterized by pathological aggregation of TDP-43 protein in ALS and either TDP-43 or Tau in FTD. One major obstacle for therapy trials in FTD is the lack of biomarkers which would allow the antemortem detection of underlying molecular pathology. Without such biomarkers, patient recruitment to TDP-43 or Tau directed therapy trials has so far been limited to rare genetic FTD cases. Here, we aimed to establish a set of blood-based biomarkers that allow the reliable distinction between patients characterized by either Tau or TDP-43 pathology.

Methods

Extracellular vesicles (EV) can transport pathological Tau and TDP-43 species between cells and induce aggregate formation in target cells. Plasma EV were prepared from 704 participants of a neurodegenerative disease cohort. Diagnostic groups included bvFTD which is largely associated with either Tau or TDP-43 pathology, Progressive Supranuclear Palsy (PSP) based on its association with 4R Tau pathology, and ALS as disorders with almost exclusive TDP-43 pathology, in addition to a healthy control group (HC).

Results

The combination of plasma EV TDP-43 and 3R/4R Tau ratios discriminated FTD cases with underlying TDP-43 from those with Tau pathology, with high sensitivity and specificity. This was confirmed by 63 cases with neuropathologically and/or genetically proven molecular pathology. High plasma EV TDP-43 levels discriminated cases with ALS, and very low Tau ratios cases with PSP, from other diagnostic groups (AUC>0.9). Both markers strongly correlated with disease severity as assessed by multiple clinical and neuropsychological scales.

Conclusions

Plasma EV Tau ratio and TDP-43 could serve as a first molecular pathology specific biomarker in ALS and FTD, which is easily accessible from blood and may have important implications for future diagnosis and therapy.

Abstract Body

Therapeutic Innovations in ALS

Amyotrophic Lateral Sclerosis is a rapidly progressive neurodegenerative disease typically leading to death within 2 – 5 years of diagnosis. Therapeutic development, until very recently, has consisted of small but significant improvements in survival and speed of functional decline. These advances and approvals are important however the goal of turning ALS into a chronic treatable condition has only recently been met is a small subset of patients, those with SOD1 mutations.
New technologies, better understanding of the underlying drivers of motor neuron cell death and the development of a biomarker driven approach is leading to real progress in treatments of ALS.
This talk will review the most current innovations leading to treatments for ALS. Therapeutic Innovations in ALS