A phase 1 proof-of-concept study evaluating safety, tolerability, and biological marker responses with combination therapy of CTLA4-Ig and interleukin-2 in amyotrophic lateral sclerosis.

Objective: To determine whether a combination therapy with abatacept (CTLA4-Ig) and interleukin-2 (IL-2) is safe and suppresses markers of oxidative stress, inflammation, and degeneration in ALS. Methods: In this open-label study, four participants with ALS received subcutaneous injections of low dose IL-2 (1 × 106 IU/injection/day) for 5 consecutive days every 2 weeks and one subcutaneous injection of CTLA4-Ig (125 mg/mL/injection) every 2 weeks coinciding with the first IL-2 injection of each treatment cycle. Participants received a total of 24 treatment cycles during the first 48 weeks in this 56-week study. They were closely monitored for treatment-emergent adverse events (TEAEs) and disease progression with the ALSFRS-R. Phenotypic changes within T cell populations and serum biological markers of oxidative stress [4-hydroxynonenal (4-HNE) and oxidized-LDL (ox-LDL)], inflammation (IL-18), and structural neuronal degeneration [neurofilament light chain (Nf-L)] were assessed longitudinally. Results: CTLA4-Ig/IL-2 therapy was safe and well-tolerated in all four participants over the 56-week study. During the first 24 weeks, the average rate of change in the ALSFRS-R was +0.04 points/month. Over the 48-week treatment period, the average rate of change was -0.13 points/month with one participant improving by 0.9 points/month while the other three participants experienced an average decrease of -0.47 points/month, which is slower than the average - 1.1 points/month prior to initiation of therapy. Treg suppressive function and numbers increased during treatment. Responses in the biological markers during the first 16 weeks coincided with minimal clinical progression. Mean levels of 4-HNE decreased by 30%, ox-LDL decreased by 19%, IL-18 decreased by 23%, and Nf-L remained the same, on average, in all four participants. Oxidized-LDL levels decreased in all four participants, 4-HNE and IL-18 levels decreased in three out of four participants, and Nf-L decreased in two out of four participants. Conclusion: The combination therapy of CTLA4-Ig and IL-2 in ALS is safe and well-tolerated with promising results of clinical efficacy and suppression of biomarkers of oxidative stress, neuroinflammation and neuronal degeneration. In this open-label study, the efficacy as measured by the ALSFRS-R and corresponding biomarkers suggests the therapeutic potential of this treatment and warrants further study in a phase 2 double-blind, placebo-controlled trial. Clinical trial registration: ClinicalTrials.gov, NCT06307301.

A phase 1 open-label pilot study of low-dose interleukine-2 immunotherapy in patients with Alzheimer's disease.

TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT05821153, Registered April 20 2023, Retrospectively registered, https://classic. CLINICALTRIALS: gov/ct2/show/NCT05821153.

Ex vivo expanded human regulatory T cells modify neuroinflammation in a preclinical model of Alzheimer's disease.

BACKGROUND: Regulatory T cells (Tregs) play a neuroprotective role by suppressing microglia and macrophage-mediated inflammation and modulating adaptive immune reactions. We previously documented that Treg immunomodulatory mechanisms are compromised in Alzheimer's disease (AD). Ex vivo expansion of Tregs restores and amplifies their immunosuppressive functions in vitro. A key question is whether adoptive transfer of ex vivo expanded human Tregs can suppress neuroinflammation and amyloid pathology in a preclinical mouse model. METHODS: An immunodeficient mouse model of AD was generated by backcrossing the 5xFAD onto Rag2 knockout mice (5xFAD-Rag2KO). Human Tregs were expanded ex vivo for 24 days and administered to 5xFAD-Rag2KO. Changes in amyloid burden, microglia characteristics and reactive astrocytes were evaluated using ELISA and confocal microscopy. NanoString Mouse AD multiplex gene expression analysis was applied to explore the impact of ex vivo expanded Tregs on the neuroinflammation transcriptome. RESULTS: Elimination of mature B and T lymphocytes and natural killer cells in 5xFAD-Rag2KO mice was associated with upregulation of 95 inflammation genes and amplified number of reactive microglia within the dentate gyrus. Administration of ex vivo expanded Tregs reduced amyloid burden and reactive glial cells in the dentate gyrus and frontal cortex of 5xFAD-Rag2KO mice. Interrogation of inflammation gene expression documented down-regulation of pro-inflammatory cytokines (IL1A&B, IL6), complement cascade (C1qa, C1qb, C1qc, C4a/b), toll-like receptors (Tlr3, Tlr4 and Tlr7) and microglial activations markers (CD14, Tyrobp,Trem2) following Treg administration. CONCLUSIONS: Ex vivo expanded Tregs with amplified immunomodulatory function, suppressed neuroinflammation and alleviated AD pathology in vivo. Our results provide preclinical evidences for Treg cell therapy as a potential treatment strategy in AD.

Combined Regulatory T-Lymphocyte and IL-2 Treatment Is Safe, Tolerable, and Biologically Active for 1 Year in Persons With Amyotrophic Lateral Sclerosis.

BACKGROUND AND OBJECTIVES: In a phase 1 amyotrophic lateral sclerosis (ALS) study, autologous infusions of expanded regulatory T-lymphocytes (Tregs) combined with subcutaneous interleukin (IL)-2 were safe and well tolerated. Treg suppressive function increased and disease progression stabilized during the study. The present study was conducted to confirm the reliability of these results. METHODS: Participants with ALS underwent leukapheresis, and their Tregs were isolated and expanded in a current Good Manufacturing Practice facility. Seven participants were randomly assigned in a 1:1 ratio to receive Treg infusions (1 × 106 cells/kg) IV every 4 weeks and IL-2 (2 × 105 IU/m2) injections 3 times/wk or matching placebo in a 24-week randomized controlled trial (RCT). Six participants proceeded into a 24-week dose-escalation open-label extension (OLE). Two additional participants entered directly into the OLE. The OLE included dose escalation of Treg infusions to 2 × 106 cells/kg and 3 × 106 cells/kg at 4-week intervals. RESULTS: The Treg/IL-2 treatments were safe and well tolerated, and Treg suppressive function was higher in the active group of the RCT. A meaningful evaluation of progression rates in the RCT between the placebo and active groups was not possible due to the limited number of enrolled participants aggravated by the COVID-19 pandemic. In the 24-week OLE, the Treg/IL-2 treatments were also safe and well tolerated in 8 participants who completed the escalating doses. Treg suppressive function and numbers were increased compared with baseline. Six of 8 participants changed by an average of -2.7 points per the ALS Functional Rating Scale-Revised, whereas the other 2 changed by an average of -10.5 points. Elevated levels of 2 markers of peripheral inflammation (IL-17C and IL-17F) and 2 markers of oxidative stress (oxidized low-density lipoprotein receptor 1 and oxidized LDL) were present in the 2 rapidly progressing participants but not in the slower progressing group. DISCUSSION: Treg/IL-2 treatments were safe and well tolerated in the RCT and OLE with higher Treg suppressive function. During the OLE, 6 of 8 participants showed slow to no progression. The 2 of 8 rapid progressors had elevated markers of oxidative stress and inflammation, which may help delineate responsiveness to therapy. Whether Treg/IL-2 treatments can slow disease progression requires a larger clinical study (ClinicalTrials.gov number, NCT04055623). CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that Treg infusions and IL-2 injections are safe and effective for patients with ALS.

Extracellular Vesicles Derived From Ex Vivo Expanded Regulatory T Cells Modulate In Vitro and In Vivo Inflammation.

Extracellular vehicles (EVs) are efficient biomarkers of disease and participate in disease pathogenesis; however, their use as clinical therapies to modify disease outcomes remains to be determined. Cell-based immune therapies, including regulatory T cells (Tregs), are currently being clinically evaluated for their usefulness in suppressing pro-inflammatory processes. The present study demonstrates that ex vivo expanded Tregs generate a large pool of EVs that express Treg-associated markers and suppress pro-inflammatory responses in vitro and in vivo. Intravenous injection of Treg EVs into an LPS-induced mouse model of inflammation reduced peripheral pro-inflammatory transcripts and increased anti-inflammatory transcripts in myeloid cells as well as Tregs. Intranasal administration of enriched Treg EVs in this model also reduced pro-inflammatory transcripts and the associated neuroinflammatory responses. In a mouse model of amyotrophic lateral sclerosis, intranasal administration of enriched Treg EVs slowed disease progression, increased survival, and modulated inflammation within the diseased spinal cord. These findings support the therapeutic potential of expanded Treg EVs to suppress pro-inflammatory responses in human disease.

Tregs Attenuate Peripheral Oxidative Stress and Acute Phase Proteins in ALS.

Oxidative stress (OS) induces inflammation, which in turn exacerbates OS and the expression of acute phase proteins (APPs). Regulatory T lymphocyte (Treg) therapy was assessed for suppression of OS and APP responses in longitudinal serum samples from subjects with amyotrophic lateral sclerosis (ALS) enrolled in a phase I clinical trial. The first round of Treg therapy suppressed levels of oxidized low-density lipoprotein (ox-LDL). During a 6-month washout period, ox-LDL levels increased. A second round of therapy again suppressed ox-LDL levels and then rose following the cessation of treatment. Serum levels of APPs, soluble CD14, lipopolysaccharide binding protein, and C-reactive protein, were stabilized during Treg administrations, but rose during the washout period and again after therapy was discontinued. Treg therapy potentially suppresses peripheral OS and the accompanying circulating pro-inflammatory induced APPs, both of which may serve as peripheral candidates for monitoring efficacies of immunomodulating therapies. ANN NEUROL 2022;92:195-200.

Serum programmed cell death proteins in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem pro-inflammatory neuromuscular disorder. Activation of programmed cell death-1 (PD-1), and its ligands, programmed cell death-ligand 1 and 2 (PD-L1/L2), leads to immune suppression. Serum soluble forms of these proteins, sPD-1/sPD-L1/sPD-L2, inhibit this suppression and promote pro-inflammatory responses. The purpose of this study was to determine if sPD-1, sPD-L1, and sPD-L2 were increased in sera of patients with ALS. sPD-1 and sPD-L2 were elevated in sera of patients and accurately reflected patients' disease burdens. Increased sera levels of programmed cell death proteins reinforce the concept that peripheral pro-inflammatory responses contribute to systemic inflammation in patients with ALS.

Ex vivo expansion of dysfunctional regulatory T lymphocytes restores suppressive function in Parkinson's disease.

Inflammation is a pathological hallmark of Parkinson's disease (PD). Chronic pro-inflammatory responses contribute to the loss of neurons in the neurodegenerative process. The present study was undertaken to define the peripheral innate and adaptive immune contributions to inflammation in patients with PD. Immunophenotyping revealed a shift of peripheral myeloid and lymphoid cells towards a pro-inflammatory phenotype. Regulatory T cells (Tregs) were reduced in number, and their suppression of T responder proliferation decreased. The PD Tregs did not suppress activated pro-inflammatory myeloid cells. Ex vivo expansion of Tregs from patients with PD restored and enhanced their suppressive functions while expanded Tregs displayed increased expression of foxp3, il2ra (CD25), nt5e (CD73), il10, il13, ctla4, pdcd1 (PD1), and gzmb. Collectively, these findings documented a shift towards a pro-inflammatory peripheral immune response in patients with PD; the loss of Treg suppressive functions may contribute significantly to this response, supporting PD as a disorder with extensive systemic pro-inflammatory responses. The restoration and enhancement of Treg suppressive functions following ex vivo expansion may provide a potential cell therapeutic approach for patients with PD.

Elevated acute phase proteins reflect peripheral inflammation and disease severity in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a multifactorial, multisystem pro-inflammatory neuromuscular disorder compromising muscle function resulting in death. Neuroinflammation is known to accelerate disease progression and accentuate disease severity, but peripheral inflammatory processes are not well documented. Acute phase proteins (APPs), plasma proteins synthesized in the liver, are increased in response to inflammation. The objective of this study was to provide evidence for peripheral inflammation by examining levels of APPs, and their contribution to disease burden and progression rates. Levels of APPs, including soluble CD14 (sCD14), lipopolysaccharide binding protein (LBP), and C-reactive protein (CRP), were elevated in sera, and correlated positively with increased disease burden and faster progression. sCD14 was also elevated in patients' CSF and urine. After a 3 year follow-up, 72% of the patients with sCD14 levels above the receiver operating characteristics cutoff were deceased whereas only 28% below the cutoff were deceased. Furthermore, disease onset sites were associated with disease progression rates and APP levels. These APPs were not elevated in sera of patients with Alzheimer's Disease, frontotemporal dementia, or Parkinson's Disease. These collective APPs accurately reflect disease burden, progression rates, and survival times, reinforcing the concept of ALS as a disorder with extensive systemic pro-inflammatory responses.

Restoring regulatory T-cell dysfunction in Alzheimer's disease through ex vivo expansion.

Inflammation is a significant component of Alzheimer's disease pathology. While neuroprotective microglia are important for containment/clearance of Amyloid plaques and maintaining neuronal survival, Alzheimer inflammatory microglia may play a detrimental role by eliciting tau pathogenesis and accelerating neurotoxicity. Regulatory T cells have been shown to suppress microglia-mediated inflammation. However, the role of regulatory T cells in ameliorating the proinflammatory immune response in Alzheimer's disease requires further investigation. Forty-six patients with Alzheimer disease, 42 with mild cognitive impairment and 41 healthy controls were studied. The phenotypes of peripheral regulatory T cells were assessed with multicolour flow cytometry. Regulatory T cells were co-cultured with responder T cells and proliferation was determined by 3H-thymidine incorporation. In separate experiments, regulatory T cells were added to induced pluripotent stem cell-derived pro-inflammatory macrophages and changes in interleukin-6/tumour necrosis-alpha transcripts and protein levels were measured. Freshly isolated regulatory T cells were expanded ex vivo in the presence of CD3/CD28 expander beads, interleukin-2 and rapamycin to promote their suppressive function. We found that the suppressive function of regulatory T cells on responder T-cell proliferation was compromised at the Alzheimer disease stage, compared with mild cognitive impairment and healthy controls. CD25 mean fluorescence intensity in regulatory T-cell population was also reduced in Alzheimer dementia patients. Regulatory T cells did not suppress pro-inflammatory macrophages at baseline. Following ex vivo expansion, regulatory T-cell suppression of responder T-cell proliferation and pro-inflammatory macrophage activation increased in both patients and controls. Expanded regulatory T cells exerted their immunoregulatory function on pro-inflammatory macrophages through a contact-mediated mechanism. In conclusion, regulatory T-cell immunophenotype and function are compromised in Alzheimer's disease. Following ex vivo expansion, the immunomodulatory function of regulatory T cells is enhanced even at advanced stages of Alzheimer's disease. Restoration of regulatory T-cell function could be explored as a means to modulate the inflammatory status of Alzheimer's disease.

Immunosuppressive Functions of M2 Macrophages Derived from iPSCs of Patients with ALS and Healthy Controls.

Amyotrophic lateral sclerosis (ALS) is a disorder with immune alterations that augment disease severity. M2 macrophages benefit diabetic and nephrotic mice by suppressing the pro-inflammatory state. However, neither have M2 cells been investigated in ALS nor have human induced pluripotent stem cell (iPSC)-derived M2 cells been thoroughly studied for immunosuppressive potentials. Here, iPSCs of C9orf72 mutated or sporadic ALS patients were differentiated into M2 macrophages, which suppressed activation of pro-inflammatory M1 macrophages as well as proliferation of ALS CD4+CD25- Tc (Teffs). M2 cells converted ALS Teffs into CD4+CD25+Foxp3+ regulatory T cells (Tregs) and rescued Tregs of ALS patients from losing CD25 and Foxp3. Furthermore, Tregs induced or rescued by iPSC-derived M2 had strong suppressive functions. ALS iPSC-derived M2 cells including those with C9orf72 mutation had similar immunomodulatory activity as control iPSC-derived M2 cells. This study demonstrates that M2 cells differentiated from iPSCs of ALS patients are immunosuppressive, boost ALS Tregs, and may serve as a candidate for immune-cell-based therapy to mitigate inflammation in ALS.

Functional alterations of myeloid cells during the course of Alzheimer's disease.

BACKGROUND: Neuroinflammation is a hallmark of neurodegenerative disease and a significant component of the pathology of Alzheimer's disease (AD). Patients present with extensive microgliosis along with elevated pro-inflammatory signaling in the central nervous system and periphery. However, the role of peripheral myeloid cells in mediating and influencing AD pathogenesis remains unresolved. METHODS: Peripheral myeloid cells were isolated from peripheral blood of patients with prodromal AD (n = 44), mild AD dementia (n = 25), moderate/severe AD dementia (n = 28), and age-matched controls (n = 54). Patients were evaluated in the clinic for AD severity and categorized using Clinical Dementia Rating (CDR) scale resulting in separation of patients into prodromal AD (CDR0.5) and advancing forms of AD dementia (mild-CDR1 and moderate/severe-CDR2/3). Separation of peripheral myeloid cells into mature monocytes or immature MDSCs permitted the delineation of population changes from flow cytometric analysis, RNA phenotype analysis, and functional studies using T cell suppression assays and monocyte suppression assays. RESULTS: During stages of AD dementia (CDR1 and 2/3) peripheral myeloid cells increase their pro-inflammatory gene expression while at early stages of disease (prodromal AD-CDR0.5) pro-inflammatory gene expression is decreased. MDSCs are increased in prodromal AD compared with controls (16.81% vs 9.53%) and have markedly increased suppressive functions: 42.4% suppression of activated monocyte-produced IL-6 and 78.16% suppression of T cell proliferation. In AD dementia, MDSC populations are reduced with decreased suppression of monocyte IL-6 (5.22%) and T cell proliferation (37.61%); the reduced suppression coincides with increased pro-inflammatory signaling in AD dementia monocytes. CONCLUSIONS: Peripheral monocyte gene expression is pro-inflammatory throughout the course of AD, except at the earliest, prodromal stages when pro-inflammatory gene expression is suppressed. This monocyte biphasic response is associated with increased numbers and suppressive functions of MDSCs during the early stages and decreased numbers and suppressive functions in later stages of disease. Prolonging the early protective suppression and reversing the later loss of suppressive activity may offer a novel therapeutic strategy.

Targeting of the class II transactivator attenuates inflammation and neurodegeneration in an alpha-synuclein model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is characterized by intracellular alpha-synuclein (α-syn) inclusions, progressive death of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and activation of the innate and adaptive immune systems. Disruption of immune signaling between the central nervous system (CNS) and periphery, such as through targeting the chemokine receptor type 2 (CCR2) or the major histocompatibility complex II (MHCII), is neuroprotective in rodent models of PD, suggesting a key role for innate and adaptive immunity in disease progression. The purpose of this study was to investigate whether genetic knockout or RNA silencing of the class II transactivator (CIITA), a transcriptional co-activator required for MHCII induction, is effective in reducing the neuroinflammation and neurodegeneration observed in an α-syn mouse model of PD. METHODS: In vitro, we utilized microglia cultures from WT or CIITA -/- mice treated with α-syn fibrils to investigate inflammatory iNOS expression and antigen processing via immunocytochemistry (ICC). In vivo, an adeno-associated virus (AAV) was used to overexpress α-syn in WT and CIITA -/- mice as a model for PD. Concurrently with AAV-mediated overexpression of α-syn, WT mice received CIITA-targeted shRNAs packaged in lentiviral constructs. Immunohistochemistry and flow cytometry were used to assess inflammation and peripheral cell infiltration at 4 weeks post transduction, and unbiased stereology was used 6 months post transduction to assess neurodegeneration. RESULTS: Using ICC and DQ-ovalbumin, we show that CIITA -/- microglial cultures failed to upregulate iNOS and MHCII expression, and had decreased antigen processing in response to α-syn fibrils when compared to WT microglia. In vivo, global knock-out of CIITA as well as local knockdown using lentiviral shRNAs targeting CIITA attenuated MHCII expression, peripheral immune cell infiltration, and α-syn-induced neurodegeneration. CONCLUSION: Our data provide evidence that CIITA is required for α-syn-induced MHCII induction and subsequent infiltration of peripheral immune cells in an α-syn mouse model of PD. Additionally, we demonstrate that CIITA in the CNS drives neuroinflammation and neurodegeneration. These data provide further support that the disruption or modulation of antigen processing and presentation via CIITA is a promising target for therapeutic development in preclinical animal models of PD.

Peripheral monocyte entry is required for alpha-Synuclein induced inflammation and Neurodegeneration in a model of Parkinson disease.

Accumulation of alpha-synuclein (α-syn) in the central nervous system (CNS) is a core feature of Parkinson disease (PD) that leads to activation of the innate immune system, production of inflammatory cytokines and chemokines, and subsequent neurodegeneration. Here, we used heterozygous reporter knock-in mice in which the first exons of the fractalkine receptor (CX3CR1) and of the C-C chemokine receptor type 2 (CCR2) are replaced with fluorescent reporters to study the role of resident microglia (CX3CR1+) and infiltrating peripheral monocytes (CCR2+), respectively, in the CNS. We used an α-syn mouse model induced by viral over-expression of α-syn. We find that in vivo, expression of full-length human α-syn induces robust infiltration of pro-inflammatory CCR2+ peripheral monocytes into the substantia nigra. Genetic deletion of CCR2 prevents α-syn induced monocyte entry, attenuates MHCII expression and blocks the subsequent degeneration of dopaminergic neurons. These results demonstrate that extravasation of pro-inflammatory peripheral monocytes into the CNS plays a key role in neurodegeneration in this model of PD synucleinopathy, and suggest that peripheral monocytes may be a target of neuroprotective therapies for human PD.

α-Synuclein fibrils recruit peripheral immune cells in the rat brain prior to neurodegeneration.

Genetic variation in a major histocompatibility complex II (MHCII)-encoding gene (HLA-DR) increases risk for Parkinson disease (PD), and the accumulation of MHCII-expressing immune cells in the brain correlates with α-synuclein inclusions. However, the timing of MHCII-cell recruitment with respect to ongoing neurodegeneration, and the types of cells that express MHCII in the PD brain, has been difficult to understand. Recent studies show that the injection of short α-synuclein fibrils into the rat substantia nigra pars compacta (SNpc) induces progressive inclusion formation in SNpc neurons that eventually spread to spiny projection neurons in the striatum. Herein, we find that α-synuclein fibrils rapidly provoke a persistent MHCII response in the brain. In contrast, equivalent amounts of monomeric α-synuclein fail to induce MHCII or persistent microglial activation, consistent with our results in primary microglia. Flow cytometry and immunohistochemical analyses reveal that MHCII-expressing cells are composed of both resident microglia as well as cells from the periphery that include monocytes, macrophages, and lymphocytes. Over time, α-Synuclein fibril exposures in the SNpc causes both axon loss as well as monocyte recruitment in the striatum. While these monocytes in the striatum initially lack MHCII expression, α-synuclein inclusions later form in nearby spiny projection neurons and MHCII expression becomes robust. In summary, in the rat α-synuclein fibril model, peripheral immune cell recruitment occurs prior to neurodegeneration and microglia, monocytes and macrophages all contribute to MHCII expression.

microRNA-155 Regulates Alpha-Synuclein-Induced Inflammatory Responses in Models of Parkinson Disease.

Increasing evidence points to inflammation as a chief mediator of Parkinson's disease (PD), a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein α-synuclein (α-syn). Recently, microRNAs, small, noncoding RNAs involved in regulating gene expression at the posttranscriptional level, have been recognized as important regulators of the inflammatory environment. Using an array approach, we found significant upregulation of microRNA-155 (miR-155) in an in vivo model of PD produced by adeno-associated-virus-mediated expression of α-syn. Using a mouse with a complete deletion of miR-155, we found that loss of miR-155 reduced proinflammatory responses to α-syn and blocked α-syn-induced neurodegeneration. In primary microglia from miR-155(-/-) mice, we observed a markedly reduced inflammatory response to α-syn fibrils, with attenuation of major histocompatibility complex class II (MHCII) and proinflammatory inducible nitric oxide synthase expression. Treatment of these microglia with a synthetic mimic of miR-155 restored the inflammatory response to α-syn fibrils. Our results suggest that miR-155 has a central role in the inflammatory response to α-syn in the brain and in α-syn-related neurodegeneration. These effects are at least in part due to a direct role of miR-155 on the microglial response to α-syn. These data implicate miR-155 as a potential therapeutic target for regulating the inflammatory response in PD.

The Metabotropic Glutamate Receptor 4 Positive Allosteric Modulator ADX88178 Inhibits Inflammatory Responses in Primary Microglia.

While the specific trigger of Parkinson Disease (PD) in most patients is unknown, considerable evidence suggests that the neuroinflammatory response makes an essential contribution to the neurodegenerative process. Drugs targeting metabotropic glutamate receptors (mGlu receptors), 7 Transmembrane (7TM) spanning/G protein coupled receptors that bind glutamate, are emerging as therapeutic targets for PD and may have anti-inflammatory properties. ADX88178 is novel potent, selective, and brain-penetrant positive allosteric modulator of the mGlu4 which is under evaluation for treatment of PD and other neurological disorders. We used microglia cultured from mouse brain to determine if ADX88178 had direct effects on the inflammatory responses of these cells. We studied both microglia from wild type and Grm4 knock out mice. We found that activation of mGlu4 with ADX88178 attenuated LPS-induced inflammation in primary microglia, leading to a decrease in the expression of TNFα, MHCII, and iNOS, markers of pro-inflammatory responses. These effects were absent in microglia from mice lacking mGlu4. These results demonstrate a cell-autonomous anti-inflammatory effect of ADX88178 mediated mGlu4 activation on microglia, and suggest that this drug or similar activators or potentiators of mGlu4 may have disease-modifying as well as symptomatic effects in PD and other brain disorders with an inflammatory component.

Fractalkine Signaling Regulates the Inflammatory Response in an α-Synuclein Model of Parkinson Disease.

BACKGROUND: Parkinson disease (PD) is a progressive neurodegenerative disorder characterized by loss of dopamine neurons in the substantia nigra pars compacta (SNpc) and widespread aggregates of the protein alpha-synuclein (α-syn). Increasing evidence points to inflammation as a chief mediator; however, the role of α-syn in triggering and sustaining inflammation remains unclear. In models of Alzheimer's disease (AD), multiple sclerosis (MS) and neurotoxin models of PD, the chemokine CX3CL1 (fractalkine) and its receptor (CX3CR1) have important roles in modulating neuroinflammation. METHODS: To examine the role of fractalkine signaling in α-syn-induced-neuroinflammation and neurodegeneration, we used an in vivo mouse model in which human α-syn is overexpressed by an adeno associated viral vector serotype 2 (AAV2) and in vitro phagocytosis and protein internalization assays with primary microglia treated with aggregated α-syn. RESULTS: We observed that loss of CX3CR1 expression led to a reduced inflammatory response, with reduced IgG deposition and expression of MHCII 4 weeks post-transduction. Six months post transduction, AAV2 mediated overexpression of α-syn leads to loss of dopaminergic neurons, and this loss was not exacerbated in animals with deletion of CX3CR1. To determine the mechanism by which CX3CR1affects inflammatory responses in α-syn-induced inflammation, phagocytosis was assessed using a fluorescent microsphere assay as well as by microglial uptake of aggregated α-syn. CX3CR1-/- microglia showed reduced uptake of fluorescent beads and aggregated α-syn. CONCLUSION: Our results suggest that one mechanism by which CX3CR1-/- attenuates inflammation is at the level of phagocytosis of aggregated α-syn by microglia. These data implicate fractalkine signaling as a potential therapeutic target for regulating inflammatory response in α-syn models PD.

MHCII is required for α-synuclein-induced activation of microglia, CD4 T cell proliferation, and dopaminergic neurodegeneration.

Accumulation of α-synuclein (α-syn) in the brain is a core feature of Parkinson disease (PD) and leads to microglial activation, production of inflammatory cytokines and chemokines, T-cell infiltration, and neurodegeneration. Here, we have used both an in vivo mouse model induced by viral overexpression of α-syn as well as in vitro systems to study the role of the MHCII complex in α-syn-induced neuroinflammation and neurodegeneration. We find that in vivo, expression of full-length human α-syn causes striking induction of MHCII expression by microglia, while knock-out of MHCII prevents α-syn-induced microglial activation, antigen presentation, IgG deposition, and the degeneration of dopaminergic neurons. In vitro, treatment of microglia with aggregated α-syn leads to activation of antigen processing and presentation of antigen sufficient to drive CD4 T-cell proliferation and to trigger cytokine release. These results indicate a central role for microglial MHCII in the activation of both the innate and adaptive immune responses to α-syn in PD and suggest that the MHCII signaling complex may be a target of neuroprotective therapies for the disease.

A partial MECP2 duplication in a mildly affected adult male: a putative role for the 3' untranslated region in the MECP2 duplication phenotype.

BACKGROUND: Duplications of the X-linked MECP2 gene are associated with moderate to severe intellectual disability, epilepsy, and neuropsychiatric illness in males, while triplications are associated with a more severe phenotype. Most carrier females show complete skewing of X-inactivation in peripheral blood and an apparent susceptibility to specific personality traits or neuropsychiatric symptoms. METHODS: We describe the clinical phenotype of a pedigree segregating a duplication of MECP2 found on clinical array comparative genomic hybridization. The position, size, and extent of the duplication were delineated in peripheral blood samples from affected individuals using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization, as well as targeted high-resolution oligonucleotide microarray analysis and long-range PCR. The molecular consequences of the rearrangement were studied in lymphoblast cell lines using quantitative real-time PCR, reverse transcriptase PCR, and western blot analysis. RESULTS: We observed a partial MECP2 duplication in an adult male with epilepsy and mild neurocognitive impairment who was able to function independently; this phenotype has not previously been reported among males harboring gains in MECP2 copy number. The same duplication was inherited by this individual's daughter who was also affected with neurocognitive impairment and epilepsy and carried an additional copy-number variant. The duplicated segment involved all four exons of MECP2, but excluded almost the entire 3' untranslated region (UTR), and the genomic rearrangement resulted in a MECP2-TEX28 fusion gene mRNA transcript. Increased expression of MECP2 and the resulting fusion gene were both confirmed; however, western blot analysis of lysates from lymphoblast cells demonstrated increased MeCP2 protein without evidence of a stable fusion gene protein product. CONCLUSION: The observations of a mildly affected adult male with a MECP2 duplication and paternal transmission of this duplication are unique among reported cases with a duplication of MECP2. The clinical and molecular findings imply a minimal critical region for the full neurocognitive expression of the MECP2 duplication syndrome, and suggest a role for the 3' UTR in mitigating the severity of the disease phenotype.