Neuronal TNFα, Not α-Syn, Underlies PDD-Like Disease Progression in IFNß-KO Mice.

OBJECTIVE: Parkinson's disease (PD) manifests in motor dysfunction, non-motor symptoms, and eventual dementia (PDD). Neuropathological hallmarks include nigrostriatal neurodegeneration, Lewy body (LB) pathology, and neuroinflammation. Alpha-synuclein (α-syn), a primary component of LBs, is implicated in PD pathogenesis, accumulating, and aggregating in both familial and sporadic PD. However, as α-syn pathology is often comorbid with amyloid-beta (Aß) plaques and phosphorylated tau (pTau) tangles in PDD, it is still unclear whether α-syn is the primary cause of neurodegeneration in sporadic PDD. We aimed to determine how the absence of α-syn would affect PDD manifestation. METHODS: IFN-ß knockout (Ifnb-/- ) mice spontaneously develop progressive behavior abnormalities and neuropathology resembling PDD, notably with α-syn+ LBs. We generated Ifnb/Snca double knockout (DKO) mice and evaluated their behavior and neuropathology compared with wild-type (Wt), Ifnb-/- , and Snca-/- mice using immunohistochemistry, electron microscopy, immunoblots, qPCR, and modification of neuronal signaling. RESULTS: Ifnb/Snca DKO mice developed all clinical PDD-like behavioral manifestations induced by IFN-ß loss. Independently of α-syn expression, lack of IFN-ß alone induced Aß plaques, pTau tangles, and LB-like Aß+ /pTau+ inclusion bodies and neuroinflammation. IFN-ß loss caused significant elevated glial and neuronal TNF-α and neuronal TNFR1, associated with neurodegeneration. Restoring neuronal IFN-ß signaling or blocking TNFR1 rescued caspase 3/t-BID-mediated neuronal-death through upregulation of c-FLIPS and lowered intraneuronal Aß and pTau accumulation. INTERPRETATION: These findings increase our understanding of PD pathology and suggest that targeting α-syn alone is not sufficient to mitigate disease. Targeting specific aspects of neuroinflammation, such as aberrant neuronal TNF-α/TNFR1 or IFN-ß/IFNAR signaling, may attenuate disease. ANN NEUROL 2021;90:789-807.

Huntingtin suppression restores cognitive function in a mouse model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in the huntingtin (HTT) protein, resulting in acquisition of toxic functions. Previous studies have shown that lowering mutant HTT has the potential to be broadly beneficial. We previously identified HTT single-nucleotide polymorphisms (SNPs) tightly linked to the HD mutation and developed antisense oligonucleotides (ASOs) targeting HD-SNPs that selectively suppress mutant HTT. We tested allele-specific ASOs in a mouse model of HD. Both early and late treatment reduced cognitive and behavioral impairments in mice. To determine the translational potential of the treatment, we examined the effect of ASO administration on HTT brain expression in nonhuman primates. The treatment induced robust HTT suppression throughout the cortex and limbic system, areas implicated in cognition and psychiatric function. The results suggest that ASOs specifically targeting mutated HTT might have therapeutic effects on HD-mediated cognitive impairments.

HACE1 is essential for astrocyte mitochondrial function and influences Huntington disease phenotypes in vivo.

Oxidative stress is a prominent feature of Huntington disease (HD), and we have shown previously that reduced levels of hace1 (HECT domain and Ankyrin repeat containing E3 ubiquitin protein ligase 1) in patient striatum may contribute to the pathogenesis of HD. Hace1 promotes the stability of Nrf2 and thus plays an important role in antioxidant response mechanisms, which are dysfunctional in HD. Moreover, hace1 overexpression mitigates mutant huntingtin (mHTT)-induced oxidative stress in vitro through promotion of the Nrf2 antioxidant response. Here, we show that the genetic ablation of hace1 in the YAC128 mouse model of HD accelerates motor deficits and exacerbates cognitive and psychiatric phenotypes in vivo. We find that both the expression of mHTT and the ablation of hace1 alone are sufficient to cause deficits in astrocytic mitochondrial respiration. We confirm the crucial role of hace1 in astrocytes in vivo, since its ablation is sufficient to cause dramatic astrogliosis in wild-type FVB/N mice. Astrogliosis is not observed in the presence of mHTT but a strong dysregulation in the expression of astrocytic markers in HACE1-/- x YAC128 striatum suggests an additive effect of mHTT expression and hace1 loss on this cell type. HACE1-/- x YAC128 mice and primary cells derived from these animals therefore provide model systems that will allow for the further dissection of Nrf2 pathways and astrocyte dysfunction in the context of HD.

Mitochondrial transcription factor A (Tfam) is a pro-inflammatory extracellular signaling molecule recognized by brain microglia.

Microglia represent mononuclear phagocytes in the brain and perform immune surveillance, recognizing a number of signaling molecules released from surrounding cells in both healthy and pathological situations. The microglia interact with several damage-associated molecular pattern molecules (DAMPs) and recent data indicate that mitochondrial transcription factor A (Tfam) could act as a specific DAMP in peripheral tissues. This study tested the hypothesis that extracellular Tfam induces pro-inflammatory and cytotoxic responses of the microglia. Three different types of human mononuclear phagocytes were used to model human microglia: human peripheral blood monocytes from healthy donors, human THP-1 monocytic cells, and human primary microglia obtained from autopsy samples. When combined with interferon (IFN)-γ, recombinant human Tfam (rhTfam) induced secretions that were toxic to human SH-SY5Y neuroblastoma cells in all three models. Similar cytotoxic responses were observed when THP-1 cells and human microglia were exposed to human mitochondrial proteins in the presence of IFN-γ. rhTfam alone induced expression of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-8 by THP-1 cells. This induction was further enhanced in the presence of IFN-γ. Upregulated secretion of IL-6 in response to rhTfam plus IFN-γ was confirmed in primary human microglia. Use of specific inhibitors showed that the rhTfam-induced cytotoxicity of human THP-1 cells depended partially on activation of c-Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (MAPK). Overall, our data support the hypothesis that, in the human brain, Tfam could act as an intercellular signaling molecule that is recognized by the microglia to cause pro-inflammatory and cytotoxic responses.

Secreted phospholipase A(2) group IIA is a neurotoxin released by stimulated human glial cells.

Neuroinflammation, which is one of the hallmarks of neurodegenerative disorders such as Alzheimer's disease, involves secretion of pro-inflammatory mediators by activated glial cells. Secreted phospholipase A(2) group IIA (sPLA(2)IIA) has been implicated as an inflammatory mediator contributing to various peripheral inflammatory conditions; however, little is known about the role this enzyme plays in neuroinflammation. Human microglia-like promonocytic THP-1 cells and human primary astrocytes were used to study sPLA(2)IIA expression, secretion and function. Production of sPLA(2)IIA by these cells was induced in response to stimulation by pro-inflammatory mediators at both mRNA and protein levels. Removal of sPLA(2)IIA from stimulated human microglia-like cell and astrocyte supernatants by immunosorbent caused significant reduction of their toxicity towards SH-SY5Y neuroblastoma cells. Both sPLA(2)IIA specific and non-specific PLA(2) inhibitors exhibited no anti-cytotoxic or neuroprotective effects, suggesting that sPLA(2)IIA cytotoxicity is mediated by a non-enzymatic mechanism. The data obtained indicate that sPLA(2)IIA may contribute to the pathogenesis of neurodegenerative diseases involving neuroinflammation. Agents inhibiting the non-enzymatic actions of sPLA(2)IIA could be used to slow down progression of neurodegenerative processes that are driven by inflammation.

Cultured adult porcine astrocytes and microglia express functional interferon-γ receptors and exhibit toxicity towards SH-SY5Y cells.

In vitro cultures of various glial cell types are common systems used to model neuroinflammatory processes associated with age-dependent human neurodegenerative diseases. Even though most researchers choose to use neonatal rodent brain tissues as the source of glial cells, there are significant variations in glial cell functions that are species and age dependent. It has been established that human and swine immune systems have a number of similarities, which suggests that cultured porcine microglia and astrocytes may be good surrogates for human glial cell types. Here we describe a method that could be used to prepare more than 90% pure microglia and astrocyte cultures derived from adult porcine tissues. We demonstrate that both microglia and astrocytes derived from adult porcine brains express functional interferon-γ receptors (IFN-γ-R) and CD14. They become toxic towards SH-SY5Y neuroblastoma cells when exposed to proinflammatory mediators. Upon such stimulation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ), adult porcine microglia, but not astrocytes, secrete tumor necrosis factor-α (TNF-α) while both cell types do not secrete detectable levels of nitric oxide (NO). Comparison of our experimental data with previously published studies indicates that adult porcine glial cultures have certain functional characteristics that make them similar to human glial cells. Therefore adult porcine glial cells may be a useful model system for studies of human diseases associated with adulthood and advanced age. Adult porcine tissues are relatively easy to obtain in most countries and could be used as a reliable and inexpensive source of cultured cells.

Synthesis and biological evaluation of novel pyrazolyl-2,4-thiazolidinediones as anti-inflammatory and neuroprotective agents.

Novel pyrazolyl-2,4-thiazolidinediones were prepared via the reaction of appropriate pyrazolecarboxaldehydes with 2,4-thiazolidinediones and substituted benzyl-2,4-thiazolidinediones. The resultant compounds were first evaluated for their anti-inflammatory and neuroprotective properties in vitro. The active compounds were further studied in vivo by using the formalin-induced paw edema and the turpentine oil-induced granuloma pouch bioassays. We identified four novel compounds that showed protective effects in vitro at non-toxic concentrations, and were also effective in the animal models of acute and sub-acute inflammation.