WO2019075094A1 - Traitement de la démence par stimulation visuelle pour induire des oscillations gama synchronisées dans le cerveau - Google Patents

Traitement de la démence par stimulation visuelle pour induire des oscillations gama synchronisées dans le cerveau Download PDF

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WO2019075094A1
WO2019075094A1 PCT/US2018/055258 US2018055258W WO2019075094A1 WO 2019075094 A1 WO2019075094 A1 WO 2019075094A1 US 2018055258 W US2018055258 W US 2018055258W WO 2019075094 A1 WO2019075094 A1 WO 2019075094A1
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visual stimuli
mice
chronic
chronic visual
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PCT/US2018/055258
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Chinnakkaruppan Adaikkan
Li-Huei Tsai
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Massachusetts Institute Of Technology
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Priority claimed from PCT/US2018/051785 external-priority patent/WO2019074637A1/fr
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Priority to JP2020520207A priority Critical patent/JP2020536643A/ja
Priority to CN201880077874.3A priority patent/CN111655319A/zh
Priority to EP18866752.1A priority patent/EP3694593A4/fr
Priority to KR1020207013291A priority patent/KR20200086277A/ko
Priority to PCT/US2018/055258 priority patent/WO2019075094A1/fr
Priority to CA3078739A priority patent/CA3078739A1/fr
Priority to KR1020237001501A priority patent/KR20230015501A/ko
Priority to AU2018347366A priority patent/AU2018347366B2/en
Publication of WO2019075094A1 publication Critical patent/WO2019075094A1/fr
Priority to AU2022271389A priority patent/AU2022271389A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
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    • A61N5/0618—Psychological treatment
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61M21/00—Other devices or methods to cause a change in the state of consciousness; Devices for producing or ending sleep by mechanical, optical, or acoustical means, e.g. for hypnosis
    • A—HUMAN NECESSITIES
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    • A61M21/00—Other devices or methods to cause a change in the state of consciousness; Devices for producing or ending sleep by mechanical, optical, or acoustical means, e.g. for hypnosis
    • A61M2021/0005—Other devices or methods to cause a change in the state of consciousness; Devices for producing or ending sleep by mechanical, optical, or acoustical means, e.g. for hypnosis by the use of a particular sense, or stimulus
    • A61M2021/0022—Other devices or methods to cause a change in the state of consciousness; Devices for producing or ending sleep by mechanical, optical, or acoustical means, e.g. for hypnosis by the use of a particular sense, or stimulus by the tactile sense, e.g. vibrations
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    • A61M21/00—Other devices or methods to cause a change in the state of consciousness; Devices for producing or ending sleep by mechanical, optical, or acoustical means, e.g. for hypnosis
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M21/00—Other devices or methods to cause a change in the state of consciousness; Devices for producing or ending sleep by mechanical, optical, or acoustical means, e.g. for hypnosis
    • A61M2021/0005—Other devices or methods to cause a change in the state of consciousness; Devices for producing or ending sleep by mechanical, optical, or acoustical means, e.g. for hypnosis by the use of a particular sense, or stimulus
    • A61M2021/0044—Other devices or methods to cause a change in the state of consciousness; Devices for producing or ending sleep by mechanical, optical, or acoustical means, e.g. for hypnosis by the use of a particular sense, or stimulus by the sight sense
    • A—HUMAN NECESSITIES
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    • A61N5/062—Photodynamic therapy, i.e. excitation of an agent

Definitions

  • AD Alzheimer's disease
  • Canter etal, 2016; Palop and Mucke, 2016 Multiple factors contribute to the pathogenesis of AD including amyloid- ⁇ deposition, hyper-phosphorylated tau accumulation, microglia- and astrocyte-mediated inflammation, and the loss of neurons and synapses (Ballatore et al, 2007; Huang and Mucke, 2012; Jacobsenefa/., 2006; Meyer-Luehmanneia/., 2008; Oakley etal, 2006; Ulelef a/., 2017; Yoshiyama et al, 2007).
  • AD pathologies such as neuronal hyper-excitability, interneuron dysfunction, shifted inhibition/excitation balance, epileptic discharges, and altered network oscillations
  • These findings are consistent with the network abnormalities observed in human AD (Guillon et al, 2017; Koenig et al, 2005; Ribary et al, 1991; Stam et al, 2002).
  • AD mouse models Recent studies in AD mouse models have highlighted that these changes occur at the presymptomatic stage (Gillespie et al, 2016; Iaccarino et al, 2016). Changes in neural activity have been previously shown to impact AD pathology, such as amyloid- ⁇ and tau accumulation, in several mouse models (Bero et al, 2011; Wu et al, 2016; Yamada et al, 2014). Given these observations, multiple approaches have been employed to investigate whether manipulating neuronal oscillations can be effective in ameliorating AD pathology (Iaccarino etal, 2016; Martinez-Losa etal, 2018; Verret eia/., 2012).
  • inventive methods and apparatus for entraining gamma oscillations in the brain of a subject via chronic non-invasive visual stimuli referred to herein as "Gamma ENtrainment Using Sensory visual stimuli” (GENUS)
  • Gamma ENtrainment Using Sensory visual stimuli has been demonstrated to extend beyond the visual cortext to multiple other brain regions (e.g., the hippocampus, somatosensory and prefrontal cortices), while also enhancing low gamma coherence across these multiple brain regions.
  • chronic GENUS reduced neurodegeneration in 5XFAD, P301S and CK-p25 mice, with the neuroprotective effect evident across multiple brain regions.
  • one inventive implementation is directed to method for treating dementia or Alzheimer's disease in a subject in need thereof, the method comprising: A) non-invasively delivering chronic visual stimuli having a frequency of about 30 Hz to about 50 Hz to the subject to entrain synchronized gamma oscillations in multiple brain regions of the subject including at least the prefrontal cortex (PFC) and the hippocampus of the subject
  • Another inventive implementation is directed to a method for treating dementia or Alzheimer's disease in a subject in need thereof, the method comprising: A) non-invasively delivering chronic visual stimuli having a frequency of about 30 Hz to about SO Hz to the subject to entrain synchronized gamma oscillations in multiple brain regions of the subject and ameliorate aberrantly modified genes and proteins in degenerating neurons in multiple brain regions of the subject.
  • Another inventive implementation is directed to a method for treating dementia or Alzheimer's disease in a subject in need thereof, the method comprising: A) non-invasively delivering chronic visual stimuli having a frequency of about 30 Hz to about SO Hz to the subject to concurrently entrain synchronized gamma oscillations in multiple brain regions of the subject to significantly increase gamma coherence having the frequency between 30 Hz to 50 Hz between the multiple brain regions of the subject.
  • the patent or application file contains at least one drawing executed in color.
  • FIGS. 1A through 1J illustrate that visual stimulation entrains gamma oscillations in multiple brain regions of a subject beyond the visual cortex, according to the inventive concepts disclosed.
  • FIGS. 2A through 2F illustrate that chronic 40 Hz (but not 80 Hz) visual flicker stimulation reduces amyloid plaques beyond visual cortex in the subject, according to the inventive concepts disclosed.
  • FIGS. 3A through 3J illustrate that chronic visual stimulation ameliorates Alzheimer's Disease-associated pathology and significantly reduces or prevents neurodegeneration in a subject, according to the inventive concepts disclosed.
  • FIGS. 4A through 4Q illustrate that chronic visual sitmulation reduces inflammatory response in microglia of a subject, according to the inventive concepts disclosed.
  • FIGS. SA through SI illustrate that chronic visual stimulation modifies synaptic function and intracellular transport in neurons, according to the inventive concepts disclosed.
  • FIGS. 6A through 61 illustrate that chronic visual stimulation modifies behavior in multiple subject models of Alzheimer's Disease, according to the inventive concepts disclosed.
  • FIGS. 7A through 71 illustrate that chronic visual stimulation entrains gamma oscillations beyond visual cortex in mouse models of neurodegeneration, according to the inventive concepts disclosed.
  • FIGS. 8A through 81 illustrate that chronic visual stimulation reduces AD-associated pathology in 5XFAD mice beyond visual cortex, according to the inventive concepts disclosed.
  • FIGS. 9A through 9G illustrate that chronic visual stimulation ameliorates AD- associated pathology in P301S and CK-p2S mice, according to the inventive concepts disclosed.
  • FIGS. 10A through 10N illustrate that chronic visual stimulation modifies microglia, improves intracellular transport and synaptic transmission in neurons, according to the inventive concepts disclosed.
  • FIGS. 11 A through 11 J illustrate behavioral characterization of the effect on a subject of acute and chronic visual stimulation according to the inventive concepts disclosed.
  • FIGS. 12A through 12C illustrate that chronic visual stimulation at 80 Hz did not affect Morris water maze in 5XFAD mice, according to the inventive concepts disclosed.
  • Gamma ENtrainment Using Sensory visual stimuli addresses symptoms related to dementia, including Alzheimer's Disease (AD), and impacts AD pathology in brain regions beyond the visual cortex.
  • AD Alzheimer's Disease
  • GENUS chronic non-invasive visual stimuli
  • chronic visual GENUS entrains gamma oscillations in multiple brain regions (including higher order brain areas) and induces functional binding at low gamma frequencies across these brain regions.
  • the visual stimuli generally had a frequency of about 30 Hz to 50 Hz, with particular attention paid to a frequency at or about 40 Hz for the visual stimuli.
  • a light-emitting diode (LED)-based device was employed to deliver the visual stimuli, and the LED-based device was driven with a square wave current pattern having a duty cycle of 50%.
  • LED light-emitting diode
  • various types of devices may be employed (other than LED-based devices) to effectively deliver the visual stimuli at various frequencies within the ranges noted herein.
  • waveforms other than square wave forms, as well as duty cycles other than 50% may be employed to effectively generate the visual stimuli at various frequencies within the ranges noted herein.
  • various treatment protocols were employed in the several studies described herein, including subject exposure times to visual stimuli of 1 hour per day, and subject exposure periods of 7 days, 22 days (about 3 weeks) and 42 days (about 6 weeks). It should be appreciated, however, that other subject exposure times and subject exposure periods may be employed to deliver visual stimuli at various frequencies within the ranges noted herein and effectively treat dementia, including Alzheimer's Disease. For example, exposure times of greater than 1 hour per day (e.g., delivered in multiple 1 hour increments, shorter increments, or longer increments), and/or exposure periods of less than three weeks, between three weeks and six weeks, and greater than six weeks, may be employed in different combinations and permuations to effectively treat dementia, including Alzheimer's Disease.
  • subject exposure times to visual stimuli of 1 hour per day e.g., delivered in multiple 1 hour increments, shorter increments, or longer increments
  • exposure periods of less than three weeks, between three weeks and six weeks, and greater than six weeks may be employed in different combinations and permuations to effectively treat dementia, including Alzheimer's
  • VI visual cortex
  • SSI somatosensory cortex
  • CA1 hippocampal area CA1
  • CC cingulate cortex
  • CA1 pyramidal cells showed strong phase-locking to low gamma (-35-45 Hz) with preferential discharging at the peak, consistent with previous reports (Bragin etal, 1995; Middleton and McHugh, 2016) (FIG. IE).
  • CK-p25 Following withdrawal of doxycycline from the diet, CK-p25 exhibit progressive neuronal and synaptic loss with cognitive impairment, which is severe by 6 weeks of p25 induction (Cruz etal., 2003). Consistent with these findings, at 6 weeks post-induction, in vivo LFP recordings from CK- p25 mice showed considerably reduced gamma spectral power at 35-45 Hz compared with control mice in VI, CA1, and PFC (FIG. 7B-7C). Despite these changes, 40 Hz visual stimulation was still able to significantly enhance gamma oscillations in VI, CA1, and PFC (FIG. 7C).
  • Average velocity was not significantly different in periods during 40 Hz visual stimulation with versus without the light occluded, negating any potential differences in locomotor activity.
  • Acute 40 Hz visual stimulation significantly increased 30 - 50 Hz low gamma coherence between visual cortex and other brain areas examined (compared with occluded light periods), specifically between VI -CA1, VI -SSI & Vl-PFC (FIG. 1G, 1H).
  • mice were exposed to 80 Hz visual stimulation, delivered with a 50% duty cycle to ensure mice received a similar light intensity and duration of exposure as in our 40 Hz stimulation experiments (FIG. II).
  • FIG. II We observed no significant changes in 80 Hz spectral power in visual cortex (where we observed the biggest increase in power with 40 Hz GENUS) during 80 Hz visual stimulation compared to pre- stimulation period (FIG. II, IS).
  • tau protein becomes hyper-phosphorylated at multiple residues (Hanger yet al, 2007; Wang et al, 2013; Foidl and Humpel, 2018; Kimura et al, 2018), and therefore utilized a non-biased Ser/Thr (S/T) phosphoproteomics approach to examine the extent to which GENUS stimulation can influence tau phosphorylation.
  • S/T residues that were hyper-phosphorylated and a single residue (S4S1) that was dephosphorylated in Tau P301S mice compared to WT naive littermates (FIG. 3C).
  • Our analysis also revealed that chronic GENUS reduced phosphorylation in the tau protein at 6 S/T sites and increased phosphorylation at S451, indicating that GENUS impacts tau phosphorylation on multiple sites (FIG. 3C).
  • cortical shrinkage is tightly correlated with ventricle expansion (Cruz etal, 2003), and we also observed a profound reduction in ventricle expansion in CK-p25 mice that received chronic GENUS (FIG. 3H, and FIG. 9E).
  • RNA sequencing revealed that microglia derived from non-stimulated CK-p25 mice, had 2333 upregulated genes when compared with CK naive mice (FIG. 4A).
  • GO gene ontology
  • the upregulated genes were involved in protein synthesis, ribosomal regulation and immune response (including viral immune-response, antigen presentation and immune response regulation), consistent with a previous report (Mathys et ah, 2017) (FIG. 4B).
  • the 2019 downregulated genes identified were primarily related to cell migration, cell morphogenesis and vasculature development (FIG. 4B).
  • CK-p25 mice with chronic GENUS showed remarkably similar transcriptomic changes as Tau P301S mice with chronic GENUS.
  • our microglia- specific transcriptomic analyses indicate that chronic GENUS acts to morphologically transform microglia, enhance protein degradation, and decrease the microglia-mediated immune response, independent of the specific transgenic model (P301S or CK-p25) to produce a disease state.
  • microglia specific marker Ibal was used to carry out immunohistochemistry and 3 -dimensional rendering, which revealed a significant increase in number and extensive changes in morphology of microglia in VI of 6- week- induced CK-p25 mice (FIG. 4D - 41 and FIG. 10E).
  • Microglia in CK-p25 animals did not show a significant difference in cell soma volume compared to controls on the whole (FIG. 4F), but many displayed a more complex 'bushy' arborization pattern (arrowheads) (FIG. 4D; lower center panel) that has been associated with axonal and terminal synaptic degeneration (Jensen et ah, 1994; Jorgensen et al, 1993).
  • microglia displayed an elongated rod-like body without polarized processes (arrow) (FIG. 4D, 41), a phenotype that is known to be present after diffuse brain injury in rats, and in human subjects with traumatic brain injury (Bachstetter et ah, 2017; Taylor et ah, 2014).
  • FIG. 4G the microglia in CK-p25 were in closer physical proximity to each other compared to CK naive controls, as analyzed by measuring the minimum distance between microglia (FIG. 4D, 4H). This indicates a loss of their territory and contrasts with resting states where each microglial cell generally has its own region of occupation, with little overlap between neighboring territories (Del Rio-Hortega, 1932; Nimmerjahn etal, 2005).
  • Chronic GENUS resulted in significantly reduced microglia numbers in CK-p25 mice compared to non-stimulated mice, although it remained higher than in naive CK mice (FIG. 4D, 4E).
  • the total volume of the microglia processes after chronic GENUS revealed less retraction, such that there was no significant difference compared to either CK-p25 and CK naive groups (FIG. 4G).
  • the minimum distance between microglia after chronic GENUS was comparable to that in CK naive animals (FIG. 4D, 4H), suggesting the preservation of microglial territories with chronic GENUS.
  • RNA-seq from these FACS -sorted neuronal RNAs. An average of 18.09 and 22.79 million reads per sample were obtained, of which 85.23% and 84.45% were aligned from CK-p25 and P301S mice, respectively. Unbiased transcriptomic analysis of NeuNf nuclei revealed that relatively more genes were downregulated in CK-p25 (618 genes) and Tau P301 S (351 genes) than upregulated genes (CK-p25: 565 genes; Tau P301 S: 229 genes), compared to their respective control mice (FIG. 5A, 5B).
  • Chronic GENUS resulted in a similar number of upregulated versus downregulated genes in CK-p25 (409 up; 422 down) and Tau P301S (220 up; 221 down), compared to their respective non-stimulated controls (FIG. 5A, 5B).
  • Downregulated genes in Tau P301 S (351 genes) were involved in chemical synaptic transmission, trans-synaptic signaling, intracellular transport including vesicle mediated transport, midbrain development and regulation of apoptotic process (FIG. SB). These same processes - including vesicle mediated transport, intracellular transport, synaptic transmission, midbrain development and regulation of apoptotic process - were all upregulated after chronic GENUS in Tau P301S mice as revealed from the top biological functions associated with the upregulated genes (FIG. SB, and FIG. 101).
  • Chronic GENUS resulted in reduced S/T phosphorylation of proteins in both CK-p25 and Tau P301S mice (FIG. 5C, 5D; bottom right panels), compared to their respective controls with no stimulation. Consistent with our neuron-specific gene expression analysis (FIG. 5A, 5B), chronic GENUS modified proteins involved in chemical synaptic transmission, dendrite development, long-term potentiation, regulation of vesicle-mediated transport, vesicle-mediated transport in synapse, and regulation of intracellular transport (FIG. SE and FIG. 10J - 10M), indicating that these processes are altered in degenerating neurons and are improved with chronic GENUS.
  • dynaminl (DNM-1) (Armbruster et al, 2013), which was associated with multiple GO terms in the annotation of differentially S/T- phosphorylated proteins after chronic GENUS in both CK-p25 and Tau P301S (FIG. 5E).
  • Phosphoregulation at Ser774 is required for endocytosis of synaptic vesicles (Clayton et al. , 2009), and is one of many residues that was found to be hyper-phosphorylated in CK-p25 and Tau P301S mice and reduced with chronic GENUS.
  • GENUS can entrain neural oscillations far beyond VI, including CA1, SSI, and PFC, and reduces AD-associated pathology including amyloid plaques, tau hyperphosphorylation, synaptic loss, and neuronal loss in all of these brain regions in CK-p25 and Tau P301S mice.
  • Our RNA-seq and phosphoproteomic data supports a capacity for chronic GENUS to mitigate some disease-associated deficits in synaptic transmission and intracellular transport that are consistent with preserved synaptic function. We therefore asked if chronic GENUS also improves cognitive function.
  • Non stimulated CK-p25 mice also exhibited impaired spatial memory as shown by reduced number of platform location visits and less time spent in the target quadrant in the probe test (24h after the last training day) compared with the CK naive group (Fischer et ah, 2005)(FIG. 6D). These impairments were significantly improved by chronic GENUS and was not the result of altered swimming velocity which remained comparable across groups and across all training days (FIG. 6D, and FIG. 11 J).
  • Locomotor activity can be a confounding factor in the detection of gamma oscillations, although we detected no difference in the velocity nor total distance travelled during the recording session between occluded and visible 40 Hz visual stimulation, making it unlikely that the low gamma entrainment we observed during GENUS is related to differences in activity levels.
  • AD is also known to cause reduced dendritic spine density and it has been shown that increasing spine density in AD mice by either genetic manipulation, pharmacological inhibition of histone deacetylases (HDACs), or optogenetics alleviates cognitive impairments (Fischer et ah, 2007; Graff et al, 2012; Roy et al, 2016).
  • HDACs histone deacetylases
  • optogenetics alleviates cognitive impairments
  • chronic visual GENUS alleviated these gene expression and protein phosphorylation defects across CK-p25 and Tau P301S models, and immunohistochemical analysis with markers specific to synaptic proteins (vGlutl, bassoon) confirmed synaptic density comparable to respective control mice.
  • AD mice, iPSC models and primary cultured cells have also implicated the disruption of intracellular transport, vesicle trafficking, and endosomal functions in AD (Millecamps and Julien, 2013; Small et al, 2017; Israel etal, 2012; Cataldo et al., 2000).
  • the link between endocytosis and ⁇ production is well documented by many studies (Marks and McMahon,1998; Cirrito et al, 2008; Schobel et a., 2008; Wu and Yao, 2009).
  • microglia phenotypes can morph as disease progresses (Mathys et ah, 2017; Lee et ah, 2018; Ulland et ah, 2017; Deczkowska et ah, 2018).
  • microglia enter a phagocytic state to uptake ⁇ in 5XFAD mice, this occurs for several modalities of GENUS (visual, auditory or a combination of both), with morphological changes in microglia correlating with a reduced amyloid plaque count (Iaccarino et al, 2016; Martorell and Paulson et al, accompanying submission). Furthermore, the phagocytic state of microglia is accompanied with an increased protein degradation (protein catabolic processes as identified by GO). The inflammatory response of microglia has recently been investigated in the context of neurodegeneration and AD.
  • FIGS. 1 A through 1J illustrate that visual stimulation entrains gamma oscillations in multiple brain regions of a subject beyond the visual cortex, according to the inventive concepts disclosed.
  • FIG. 1A 40 Hz visual stimulation with 50 % duty cycle (12.5 ms lights on and 12.5 ms lights off) was delivered using an chicken system.
  • C57BL/6J mice received either no stimulation or 40 Hz visual stimulation for a 1 h period, after which they were sacrificed and brains stained for c-Fos expression.
  • Microdrives were implanted in a separate cohort for in vivo electrophysiology.
  • group gamma area power (40 ⁇ 5 Hz) was calculated from FIG. 1 C.
  • FIG. IE we implanted custom-made tetrode microdrive in C57BL/6J mice, tetrodes were adjusted to CA1 and single units were isolated. Chart shows the spike probability across 40 Hz phase in light occluded and 40 Hz stimulation periods.
  • WPLI weighted phase lag index method
  • FIG. 11 is a schematic of 80 Hz LED light delivery with 50 % duty cycle (6.25 ms lights on and 6.25 ms lights off).
  • FIG. 1 J shows power spectra of VI LFP in C57B1/6J mice subjected to light occluded (Oc. LED) or 80 Hz visual stimulation.
  • FIGS. 2A through 2F illustrate that chronic 40 Hz (but not 80 Hz) visual flicker stimulation reduces amyloid plaques beyond visual cortex in the subject, according to the inventive concepts disclosed.
  • FIG. 2A shows amyloid plaque load in 5XFAD mice exposed to either no stimulation, 40 Hz or 80 Hz visual stimulation lh per day for 7 days, as visualized by immunohistochemistry staining of the D54D2 antibody.
  • FIG. 2B illustrates group quantification showing that GENUS lh per day for 7 days reduced amyloid plaques in VI, but it did not significantly alter amyloid plaques in SSI or hippocampal area CA1.
  • 80 Hz visual stimulation exposure did not alter levels of amyloid plaques in VI or CA1 , whereas it significantly increased amyloid plaques in the SS 1.
  • N 8 non-stimulated, and 6 mice each from 40 Hz and 80 Hz group.
  • FIG. 2C shows representative images of amyloid plaque load in 5XFAD mice exposed to either no stimulation or an extended GENUS protocol of 22 days (lhour per day). Scale bars represent 50 ⁇ .
  • FIG. 2D shows that 22 days of GENUS significantly reduced amyloid plaques in VI, SSI , CAl and CC.
  • N 6 mice per condition.
  • Two-way ANOVA between groups effect F (1 , 40) 51.00, P ⁇ 0.0001. Bonferroni's multiple comparisons test, *** P ⁇ 0.001, ** P ⁇ 0.01.
  • FIG. 2E shows representative images of amyloid plaques in VI , SS 1 and CAl visualized from mice exposed to no stimulation or 80 Hz light flicker for lh per day for 22 days, scale bar represents 50 ⁇ .
  • FIG. 2F is related to FIG. 2E, showing group data quantifying amyloid plaques in VI, SSI and CAl.
  • FIGS. 3A through 3J illustrate that chronic visual stimulation ameliorates Alzheimer's Disease-associated pathology and significantly reduces or prevents neurodegeneration in a subject, according to the inventive concepts disclosed.
  • FIG. 3 A provides an experiment outline showing that P301 S mice were subjected to no stimulation or GENUS for 22 days (lh per day) followed by immunohistochemical and phosphoproteomics analyses. WT cage littermates that did not undergo stimulation regimen were considered WT naive mice.
  • FIG. 3C illustrates phosphoproteomic analysis of Serine/Threonine phosphorylated tau protein from visual cortex.
  • Statistical comparison (P values) between non-stimulated and GENUS stimulated P301S mice for specific residues are shown in the chart.
  • GENUS reduced phosphorylation in the tau protein at 6 S/T sites and increased phosphorylation at S451. Commonly known human tau S/T sites are shown on top.
  • FIG. 3D shows representative images for the neuronal marker NeuN in visual cortex from WT naive and P301S mice that underwent no-stimulation or GENUS. Scale bar represents 50 ⁇ .
  • FIG 3E shows group data indicating P301 S mice showed significant loss of neurons in VI, SSI, CAl and CC compared to WT naive mice.
  • GENUS stimulated P301S group showed significantly reduced neurodegeneration.
  • N same as in FIG. 3B.
  • Two-way ANOVA between groups effect F (2, 76) 19.73, P ⁇ 0.0001.
  • FIG. 3F provides and experimental overview showing induction of p25 expression in CK-p25 mice for 42 days. This was accompanied by GENUS for lh per day in one experimental group, the non-stimulation control group received room light.
  • CK (CaMK2a-promoter x tTA) cage littermates that did not undergo stimulation regimen were considered CK naive mice.
  • FIG. 3G is a photomicrograph showing qualitative differences in brains between CK- naive, non-stimulated and GENUS stimulated CK-p25 mice, 42 days post induction.
  • CK- p25 mice exhibited reduced brain weight (i.e. brain atrophy) compared to CK naive mice, whereas chronic GENUS partially alleviated brain atrophy in CK-p25 mice.
  • N 13 CK naive mice, 10 mice each for non-stimulated and GENUS CK-p25 groups.
  • ANOVA F (2, 30) 15.46, P ⁇ 0.001. Post-hoc multiple comparisons with Bonferroni's correction, **** P ⁇ 0.0001, * P ⁇ 0.05.
  • FIG. 31 provides representative images for the neuronal marker NeuN in visual cortex from CK naive and CK-p25 mice that underwent no-stimulation or GENUS. Scale bar represents 50 ⁇ .
  • FIG. 3J shows that CK-p25 mice exhibited severe neuronal loss in VI, SSI, CA1 and CC, whereas chronic GENUS reduced the loss of neurons in CK-p25 mice.
  • N same as in FIG. 3G.
  • Two-way ANOVA F (2, 72) 31.38, P ⁇ 0.0001. Post-hoc multiple comparisons with Bonferroni's correction **** P ⁇ 0.001, *** P ⁇ 0.001, ** P ⁇ 0.01, * P ⁇ 0.05.
  • FIGS. 4A through 4Q illustrate that chronic visual sitmulation reduces inflammatory response in microglia of a subject, according to the inventive concepts disclosed.
  • FACS fluorescence activated cell sorting
  • FIG. 4B shows selected gene ontology (GO) terms for biological processes associated with the identified DEGs. Top: GO terms associated with the upregulated (UP) genes in non- stimulated CK-p25 mice compared to CK naive mice. Bottom: GO terms associated with the downregulated (DOWN) in non-stimulated CK-p25 mice compared to CK naive mice.
  • FIG. 4C shows selected GO terms associated with DEGs. Top. GO terms associated with the upregulated (UP) genes in GENUS CK-p25 mice compared to non-stimulated CK-p25 mice. Bottom: GO terms associated with the downregulated (DOWN) in GENUS CK-p25 mice compared to non-stimulated CK-p25 mice.
  • FIG. 4D provides representative images showing microglia marker Ibal (green) and CD40 (red) immunostaining from visual cortex. Scale bar 50 ⁇ .
  • N 7 CK naive mice, 6 mice each for non-stimulated and GENUS groups. Arrow and arrowhead indicate rod-shaped ramified processes and branch volume of microglia processes respectively.
  • FIG. 4E shows that non-stimulated CK-p25 mice exhibited higher number of Ibal positive cells compared to CK naive mice, chronic GENUS significantly reduced Ibal cell density in CK-p25 mice.
  • ANOVA F (2, 16) 17.79, P ⁇ 0.0001.
  • FIG. 4F we performed three-dimensional rendering of microglia using Imaris and quantified the volume of soma and processes of microglia.
  • N 73 microglia per group from the same number of mice as in FIG. 4D.
  • FIG. 4G shows the overall volume of microglia processes (excluding rod like microglia) was significantly lower in non-stimulated CK-p25 mice compared to CK naive mice.
  • FIG. 4H illustrates quantification of minimum distance between microglia.
  • N 68 microglia from 9 CK naive, 131 microglia from 6 non-stimulated and 95 microglia from 6 GENUS CK-p25 mice.
  • Microglia in non-stimulated CK-p25 mice aggregated together compared to CK naive mice, whereas GENUS significantly reduced microglia aggregation (H 100.1, P ⁇ 0.0001).
  • FIG. 41 illustrates quantification of radial primary processes of rod like microglia.
  • N 16 microglia from 9 CK naive, 19 microglia per group from 6 mice each from non-stimulated and GENUS CK-p25 group.
  • the overall volume of processes of rod like microglia was significantly reduced after GENUS in CK-p25 mice compared to non-stimulated CK-p25 mice.
  • ANOVA F (2, 51) 16.27, P ⁇ 0.0001.
  • FIG. 4J shows that non-stimulated CK-p25 mice exhibited higher signal intensity of interferon response protein CD40 compared to CK naive mice, whereas chronic GENUS significantly reduced CD40 signal in CK-p25 mice.
  • ANOVA F (2, 16) 36.84, P ⁇ 0.0001.
  • FIG. 4K provides representative images from visual cortex showing Ibal in green and nuclear stain Hoechst in blue. Scale bar 50 ⁇ . Arrowhead indicates the complexity of microglia processes.
  • FIG. 4L illustrates that P301 S tau mice showed a trend towards increased total number of Ibal positive cells but was not statistically significant compared to WT mice.
  • N 7 WT naive, 8 non-stimulated P301S mice and 7 GENUS mice.
  • FIG. 4M is a frequency distribution chart showing the size of microglia soma.
  • N 7 WT naive, 8 non-stimulated P301S mice and 7 GENUS mice. A total of 58 microglia per group was analyzed. The overall volume of microglia soma did not differ between groups.
  • FIG. 4N shows that the volume of the processes of microglia was smaller in P301 S tau mice compared to WT mice, whereas GENUS stimulated P301S mice showed similar process length to that of WT mice.
  • FIG. 4P shows that non-stimulated CK-p25 mice exhibited higher Clq signal intensity compared to CK naive mice, whereas chronic GENUS stimulated CK-p25 mice showed significantly reduced Clq intensity.
  • FIG. 4Q shows that non-stimulated P301S mice exhibited higher Clq signal intensity compared to WT naive mice, chronic GENUS did not differ between any groups.
  • FIGS. 5A through 51 illustrate that chronic visual stimulation modifies synaptic function and intracellular transport in neurons, according to the inventive concepts disclosed.
  • CK-p25 mice were subjected to no stimulation or GENUS lh per day for 42 days.
  • NeuN positive (100,000) nuclei of neurons from visual cortex were isolated using FACS followed by RNA extraction and sequencing.
  • Heat-maps of the DEGs Number of mice per group is indicated in the heat-maps.
  • Right Chart showing the biological processes associated with the downregulated genes in non-stimulated CK-p25 mice compared to CK naive mice, and the overlap of the same biological process associated with the upregulated genes after GENUS in CK-p25 mice.
  • FIG. 5B P301 S mice were subjected to no stimulation or GENUS stimulation lh per day for 22 days.
  • NeuN positive (100,000) nuclei of neurons from visual cortex were isolated using FACS followed by RNA extraction and sequencing. Heat-maps of the differentially expressed genes. Number of mice per group is indicated in the heat-maps. Top left: DEGs between WT naive and non-stimulated P301S tau mice. Bottom left. DEGs between non-stimulated and GENUS stimulated P301S tau mice, number of genes is indicated on the right. Right. Chart showing the biological processes associated with the downregulated genes in non-stimulated P301S mice compared to CK naive mice, and the overlap of the same biological process associated with the upregulated genes after GENUS stimulation in P301S mice.
  • FIG. 5D P301 S tau mice were subjected to no stimulation or GENUS stimulation for 22 days.
  • Total protein expressions and S/T phosphorylated proteins analysis was performed on visual cortex tissue using tandem mass tag (TMT) 10-plex kit (see methods) and mass spectrometry (LC-MS/MS).
  • TMT tandem mass tag
  • LC-MS/MS mass spectrometry
  • N 3 WT naive, 3 non-stimulated P301S and 4 GENUS stimulated P301S mice.
  • Top Venn diagram showing the overlap of total RNAs identified from neuron specific RNA seq (FIG. 5A, above) and total proteins identified from LC-MS/MS. 91.95% of the proteins identified were found to be expressed in neurons.
  • FIG. 5E shows GO terms for the differentially S/T phosphorylated proteins in CK-p25 and P301 S tau mice after chronic GENUS.
  • FIG. 5F shows representative images showing neurofilament heavy chain (NFH, a neuronal marker expressed primarily in axons) and Ser774 phosphorylated dynaminl immunostaining from visual cortex.
  • NFH neurofilament heavy chain
  • SA774 phosphorylated dynaminl immunostaining was used to label neuronal processes.
  • N 7 CK naive mice, 6 mice each for non-stimulated and GENUS stimulated groups.
  • Middle CK-p25 mice exhibited significantly higher levels of pS774-DNMl signal intensity compared to CK naive, whereas GENUS significantly reduced this aberrant phosphorylation in CK-p25 mice.
  • F (2, 16) 38.551, P ⁇ 0.0001.
  • 5G shows representative brain slices immunostained for vesicular glutamate transporter 1 (vGlutl) from visual cortex of CK naive, non-stimulated and GENUS stimulated CK- p25 mice.
  • Scale bar represents 10 ⁇ .
  • FIG. 5H shows that while the expression of vGlutl puncta was significantly lower in non-stimulated CK-p25 mice, GENUS stimulated CK-p25 mice exhibited higher levels of vGlutl puncta compared to non-stimulated CK-p25 mice in VI, SSI , CA1 and CC.
  • N 9 CK naive mice, and 6 mice each from non-stimulated and GENUS CK-p25 group.
  • Two-way ANOVA between groups effect F (2, 72) 42.06, P ⁇ 0.0001.
  • FIG. 51 shows that the expression of synaptic puncta vGlutl was significantly lower in P301S compared to WT naive mice, GENUS rescued vGlutl expression in VI , CA1 and CC in P301S.
  • N 7 WT naive, and 8 non-stimulated P301S mice and 7 GENUS mice.
  • Two-way ANOVA between groups effect F (2, 76) 23.67, P ⁇ 0.0001.
  • FIGS. 6A through 61 illustrate that chronic visual stimulation modifies behavior in multiple subject models of Alzheimer's Disease, according to the inventive concepts disclosed.
  • FIG. 6A p25 expression was induced in two groups of CK-p25 mice for 42 days, only one of which received daily GENUS for 1 h (stimulation was performed between 9 am- 12 pm). Mice were subjected to open field (OF) and novel object recognition (OR) tests on day 40 (OF) and day 41 (OR) afternoon (3 pm-7 pm). F and N denote familiar and novel objects, respectively. Representative occupancy heat maps from OF and OR sessions are shown. Color level is mapped to the range of location frequencies in the arena or objects: warm color represents higher time and frequency, whereas cold color represents less time in the specified locations.
  • FIG. 6B shows that chronic visual GENUS did not affect anxiety levels in CK-p25 mice.
  • Time spent in the center of OF arena did cold not differ between CK naive, non-stimulated and GENUS stimulated CK-p25 mice.
  • FIG. 6C shows preference for novel objects in CK naive and GENUS stimulated CK- p25 mice was significantly higher than familiar object, however; non-stimulated CK-p25 mice showed no preference.
  • FIG. 6D shows Morris water maze performance of p25 induced CK-p25 mice with or without GENUS for 42 days (MWM; between day 36 and 41 of p25 induction).
  • Top Chronic GENUS reduced the latency to find the platform in the training in CK-p25 mice compared to non- stimulated CK-p25 mice.
  • Two-way ANOVA between groups effect F (2, 252) 18.64, P ⁇ 0.0001.
  • FIG. 6E relates to P301 S tau mice exposed to no stimulation or GENUS for lh per day for 22 days (9 am- 12pm) then assessed in OF (day 20; 3 - 7 pm) and OR (day 21) tests. Representative occupancy heat maps from OF and OR sessions are shown.
  • FIG. 6G shows that WT, non-stimulated and GENUS stimulated P301 S mice all exhibited higher preference for novel objects.
  • FIG 6H shows MWM performance of WT naive, non-stimulated or GENUS stimulated P301 S tau mice.
  • FIG. 61 5XFAD mice with or without GENUS (lh per day for 22 days) were tested for MWM performance.
  • FIGS. 7A through 71 illustrate that chronic visual stimulation entrains gamma oscillations beyond visual cortex in mouse models of neurodegeneration, according to the inventive concepts disclosed.
  • FIG. 7A shows electrolytic lesions to verify recording sites in mice used for main figures 1C, ID post- recording. Representative images show the site of recording from VI, SSI, CA1 and PFC.
  • CDK5 activator p25 was induced in CK-p25 mice for 6 weeks, followed by microdrive implantation and LFP recording.
  • FIG. 7D microdrives were implanted in 8 month old P301 S tau mice.
  • FIG. 7F relates to C57B16/J mice subjected to no stimulation or GENUS 1 h/day for 42.
  • FIG. 71 shows that significant increases were also evident in low gamma coherence between VI- CA1 (P ⁇ 0.01), CA1-PFC (P ⁇ 0.01), Vl-PFC (P ⁇ 0.01).
  • FIGS. 8A through 81 illustrate that chronic visual stimulation reduces AD-associated pathology in 5XFAD mice beyond visual cortex, according to the inventive concepts disclosed.
  • FIG. 8A is a schematic showing the visual flicker stimulation set up.
  • An array of light emitting diodes (LEDs) was present on the open side of the cage and was driven to flicker at a frequency of 40 Hz with a square wave current pattern using an electrician system. Since mice freely move in the cage (83 in 2 total floor area), the light intensity that they received ranges from ⁇ 1500 to 300 lux.
  • FIG 8B 10 month old 5XFAD mice underwent no stimulation or GENUS lh pea- day for 22 days. Representative images show amyloid plaques in red and nuclear stain Hoechst in blue from slices containing hippocampus and somatosensory cortex. Scale bar 1000 ⁇ . Related to main figure 2C- 2D.
  • FIG 8C 10 month old 5XFAD mice underwent no stimulation or GENUS lh pea- day for 22 days. Representative images show amyloid plaques in red, neuronal marker NeuN in green and nuclear stain Hoechst in blue from cingulate cortex. Scale bar 50 ⁇ . Related to main figure 2C- 2D.
  • FIG. 8D shows that the number of plaques in GENUS stimulated 5XFAD mice was significantly lower than non-stimulated 5XFAD mice in VI, SSI, CA1 and CC.
  • N 6 mice per condition.
  • Two-way ANOVA between groups effect F (1, 40) 30.01, P ⁇ 0.0001.
  • FIG. 8F 10 month old 5XFAD mice underwent no stimulation or GENUS 1 h per day for 22 days. Representative images show synaptic marker bassoon. Scale bar 10 ⁇ .
  • FIG 8H provides full length immunoblots showing the expression levels of full length APP protein and endogenous GAPDH control.
  • N 5 WT naive mice, 3 non-stimulated 5XFAD mice and 4 GENUS stimulated 5XF AD mice.
  • FIG. 81 shows that APP protein expression was significantly higher in 5XFAD mice compared to WT naive control. Between non-stimulated and GENUS stimulated 5XFAD mice it did not differ. Note that we did not measure the C/N fragments of APP proteins.
  • FIGS. 9A through 9G illustrate that chronic visual stimulation ameliorates AD- associated pathology in P301S and CK-p25 mice, according to the inventive concepts disclosed.
  • FIG 9A provides full length immunoblots showing the expression levels of total tau protein and GAPDH from visual cortex of non-stimulated, GENUS stimulated P301S tau mice, and age matched WT naive littermates.
  • Total tau expression in P301S tau mice was increased compared to WT naive mice, however; tau levels did not differ between non-stimulated and GENUS stimulated P301S mice.
  • N 3 WT naive, 3 non-stimulated and 4 GENUS stimulated P301S mice.
  • ANOVA F (2, 7) 173.275, P ⁇ 0.0001. Bonferroni's multiple comparisons test, ***P ⁇ 0.001.
  • FIG. 9C provides full length immunoblots showing the expression levels of p25 +GFP (fusion protein), p25, p35 and GAPDH from visual cortex of CK naive, non-stimulated and GENUS stimulated CK-p25 mice.
  • p25 +GFP fusion protein
  • p25, p35 and GAPDH from visual cortex of CK naive, non-stimulated and GENUS stimulated CK-p25 mice.
  • FIG. 9E provides representative images showing the qualitative differences among CK naive, non-stimulated and GENUS stimulated CK-p25 mice detailing changes in hippocampal volume, cortical thickness and ventricle size.
  • FIG. 3F - 3H provides representative images showing the qualitative differences among CK naive, non-stimulated and GENUS stimulated CK-p25 mice detailing changes in hippocampal volume, cortical thickness and ventricle size.
  • FIG. 9F shows that GENUS did not alter the p25 +GFP fusion protein expression level (number of p25: GFP expressing neurons) compared to non-stimulated CK-p25 mice, across all brain regions tested, independent samples t-test between groups, P > 0.05.
  • FIGS. 10A through 10N illustrate that chronic visual stimulation modifies microglia, improves intracellular transport and synaptic transmission in neurons, according to the inventive concepts disclosed.
  • FIG. 10A shows microglia separation profile in FACS from representative CK naive, non-stimulated and GENUS stimulated CK-p25 mice.
  • FIG. 4A 4C shows microglia separation profile in FACS from representative CK naive, non-stimulated and GENUS stimulated CK-p25 mice.
  • FIG. 10B shows microglia separation profile in FACS from representative WT naive, non-stimulated and GENUS stimulated P301S tau mice.
  • DEGs differentially expressed genes
  • FIG. 10D shows the top 7 processed gene ontology (GO) terms for biological processes associated with the identified DEGs. Group comparisons are shown to the top.
  • GO gene ontology
  • FIG. 10E provides representative images showing Ibal in green and CD40 in red. Merged images presented in main FIG. 4D were separated for clarity.
  • FIG. 10F shows neuronal nuclei separation profile in FACS from representative CK naive, non-stimulated and GENUS stimulated CK-p25 mice is shown.
  • Group mean and SEM of % NeuN compare to total nuclei: CK naive, 50 ⁇ 1.48; non-stimulated CK-p25, 40.98 ⁇ 0.719; GENUS stimulated CK-p25 mice, 45.08 ⁇ 1.691.
  • FIG. 10G shows neuronal nuclei separation profile in FACS from representative WT naive, non-stimulated P301S and GENUS stimulated P301S mice is shown.
  • FIG. 10H provides bar graphs showing the percentage of total neurons (NeuN) positive nuclei compare to total nuclei as in FIG. 10F and 10G.
  • FIG. 101 upregulated genes in P301S tau mice and CK-p25 mice after chronic GENUS were selected based on neurotransmitter transport and vesicle mediated transport clusters with 60 genes. Note that these genes were upregulated only in CK-p25, P301S or in both mice after GENUS compared to non-stimulated mice (analyzed by metascape). Protein-protein interaction network map (Analyzed with Cytoscape V3.6.1 followed by Strings; enrichment P value, 6.66E-15) is shown with tightly linked further functional enrichment GO terms.
  • FIG. 10J the visual cortex from non-stimulated P301S tau-tg mice and GENUS stimulated lh/d for 22 days were subjected to S/T phosphoproteomics analysis.
  • Heat-map shows the differentially phosphorylated proteins between non-stimulated and GENUS stimulated P301S mice that are involved in synaptic transmission and intracellular transport
  • FIG. 10K a heat-map shows the differentially phosphorylated proteins involved in synaptic transmission (chemical synaptic transmission, trans-synaptic signaling) between non- stimulated and GENUS stimulated CK-p25 mice. Genes (corresponding proteins) names and the specific phospho-sites are shown.
  • FIG. 10L P301S tau mice were subjected to no stimulation or GENUS stimulation for 22 days.
  • Total protein expressions and S/T phosphorylated proteins analysis was performed on visual cortex tissue using tandem mass tag (TMT) 10-plex kit (see methods) and mass spectrometry (LC-MS/MS).
  • TMT tandem mass tag
  • LC-MS/MS mass spectrometry
  • N 3 WT naive, 3 non-stimulated P301S and 4 GENUS stimulated P301S mice.
  • Phosphorylated proteins with fold change of ⁇ 0.2 and adjusted P value of ⁇ 0.05 were considered statistically significant GO terms of biological processes associated with the differentially S/T phosphorylated proteins in CK-p25 and P301S tau mice compared to their respective control mice.
  • FIG. 10M - Top Western blots show pS774 DNM-1, DNM-1, DNM-3 and GAPDH from CK naive, non-stimulated and 40 Hz GENUS stimulated CK-p25 mice.
  • pS774DNM-l level was significantly higher in CK-p25 mice compare to CK naive mice, whereas 40 Hz GENUS significantly reduced pS774DNM-l in CK-p25 mice.
  • Bottom Bar chart shows the group difference. Please note that this is an independent verification.
  • FIG. 5F Western blots show pS774 DNM-1, DNM-1, DNM-3 and GAPDH from CK naive, non-stimulated and 40 Hz GENUS stimulated
  • FIG 10N - Top Western blots show pS774 DNM-1, total DNM-1, DNM-3 and GAPDH from WT naive, non-stimulated and 40 Hz GENUS stimulated P301S mice. Please note that this is an independent verification. *P ⁇ 0.05. Related to main FIG. 5F.
  • FIGS. 11 A through 11J illustrate behavioral characterization of the effect on a subject of acute and chronic visual stimulation according to the inventive concepts disclosed.
  • FIG. 1 ID C57B16/J mice underwent no stimulation or GENUS 1 h per day for 7 days.
  • the body weight of mice was measured lh before the stimulation paradigm every day for 7 days and 1-day post stimulation regimen.
  • N 13 WT naive, 14 non-stimulated and 12 GENUS stimulated P301S mice.
  • N 25 CK naive, 21 non-stimulated and 18 GENUS stimulated CK-p25 mice.
  • FIG. 1 II - tefi Plasma corticosterone levels did not differ between non-stimulated and 7 days GENUS stimulated WT mice.
  • N 12 mice per group.
  • Middle Mice underwent no stimulation or GENUS lh per day for 42 days while p25 was induced in CK-p25 mice. Number of mice per group is indicated in the chart.
  • FIG 11 J is related to main FIG 7.
  • Left No differences between any groups across all 6 days of training in CK-p25 mice were evident (FIG. 7D).
  • Middle There was no difference between groups across all 5 days of training in P301S tau mice (FIG. 7H).
  • Two-way ANOVA between groups effect F (2, 273) 24.24, PO.0001.
  • FIGS. 12A through 12C illustrate that chronic visual stimulation at 80 Hz did not affect Morris water maze in 5XFAD mice, according to the inventive concepts disclosed.
  • FIG. 12B indicates the number of platform crossings during the probe test.
  • FIG 12C shows time spent in the target quadrant during the probe test was significantly less in the non-stimulated and 80 Hz stimulated 5XFAD mice compared to WT nai ve mice.
  • mice used were 3, 10 or 17 months old.
  • CK-control and CK-p25 (Camk2a- tTA bred with tetO-CDK5Rl/GFP) mice were raised on a doxycycline containing food. Normal rodent diet was given to induce p25-GFP transgene expression. Typically, p25 expression was induced for 6 weeks.
  • P301 S tau mice were 7 months and 5XFAD mice were 9 or 11 months. All mice were group housed (2- 5 mice per cage) except those that were implanted with microdrives. All experiments were done using age matched littermates.
  • mice After lh of 40 Hz light flicker exposure mice were returned to their home cage and allowed to rest for a further 30 minutes before being transported back to the holding room. Normal room light control mice were exposed to the similar cages with similar food and water restriction, however; they experienced only normal room light.
  • NeuN, c-Fos and GFP positive cell counting All images were acquired in Z-stacks- 10 per image and were quantified. An average of every two and sum of all the counts was computed using ImageJ. NeuN counting from P301S tau-tg and 5XFAD was done by an experimenter blind to the treatment condition.
  • vGlutl, bassoon puncta LSM 880, with a 63 x objective and further zoom of 3 times was used to acquire the images.
  • the particle count plugin in ImageJ was used to quantify the number of vGlutl puncta.
  • pS202/T205 tau intensities Using a LSM 710 with a 40* objective z-stacks of the entire slice thickness 40 ⁇ (40 images from each field) were acquired. All images were compressed/collapsed and the signal intensity was measured in ImageJ.
  • Plaques The D54D2 antibody stained overall plaque intensity and the number of plaques was quantified by an experimenter blind to the genotype and treatment conditions. The entire 40 ⁇ slice was z-stack imaged at 0.S ⁇ intervals, these were then merged and the signal intensity measured using ImageJ. Plaques were counted using the particle analysis tool in ImageJ with a threshold of 5 ⁇ 2 . All plaque intensities and plaque number counting were done by an experimenter blind to the treatment condition.
  • Clq intensities Using a LSM 710 with a 40x objective z-stacks of the entire slice thickness 40 ⁇ (40 images from each field) were acquired. All images were compressed/collapsed and the signal intensity was measured in ImageJ.
  • CD40 An LSM 880 with a 63 * objective was used to obtain z-stacks of the entire slice thickness 40 ⁇ (40 images from each field). All images were compressed/collapsed and the signal intensity was measured in ImageJ.
  • Lateral ventricles LSM 710 microscope with a 5x objective was used to image complete coronal slices at -2.0 bregma (rostral to caudal). Outlines covering the entire area of the lateral ventricles were drawn using the freehand selection tool from ImageJ and the area of the LV was measured.
  • Microglia Ibal immunoreactive cells were considered microglia. Using a LSM 710 with a 40x objective z-stacks of the entire slice thickness 40 ⁇ (40 images from each field) were acquired. Imaris was used for 3D rendering of images to quantify the total volume of soma and processes microglia. Ibal aggregation analysis was performed in Image J 3D rendering plugin. Minimum distance between Ibal was calculated for every microglia from the images. All images were compressed/collapsed and Image J was used to quantify the total number of Ibal positive cells.
  • Cortical thickness LSM 710 microscope with a 5X objective was used to image complete cortical columns. The distance between outer cortical boundary and cortical side of corpus callosum was measured using ImageJ.
  • Brain weight measurement Mice were transcardially perfused with PBS followed by 4% PFA and the brain was post-fixed overnight in 4% PFA. Brains were washed in PBS and any excess removed before being weighed in a wet lab high precision scale (Mettler Toledo, accurate to 1 mg). Another cohort of mice were sacrificed, brain was flash frozen in liquid nitrogen and brain weight was measured. Brain weight was normalized to CK naive brains in each of these two independent measures.
  • Tungsten wire electrode drives Microdrives were custom built using a 3D printed drive base with perfluoroalkoxy coated tungsten wire electrodes (50 ⁇ bare diameter, 101.6 coated diameter; A-M Systems) and Neuralynx electrode interface board (EIB-36). Polyimide tubes were used to protect electrodes and reduce electrical noise.
  • Electrodes were arranged to target layer 3 or 4 of visual cortex (co-ordinates relative to bregma, anterior-posterior (AP), -3.0; medial-lateral (ML), + 2.0), SSI (AP, -2.0; ML, + 2.3), CAl region of the hippocampus (AP, -1.8; ML, +1.5) and cingulate area of the prefrontal cortex (AP, +1.0; ML, +0.2).
  • Reference electrode was placed in the cerebellum.
  • Tetrode drives Custom microdrives contain four nichrome tetrodes (14 mm; California Fine Wire Company), gold-plated (Neuralynx) to an impedance of 200 to 250 kU, arranged in one row (running along the CA3 to CAl axis of the dorsal hippocampus) was implanted to dorsal CAl (AP, -1.8). Reference electrode was placed in the fiber tract above the hippocampus.
  • Spikes Single units was manually isolated by drawing cluster boundaries around the 3D projection of the recorded spikes, presented in SpikeSort3D software (Neuralynx). Cells were considered pyramidal neurons if mean spike width exceeded 200 ⁇ as and had a complex spike index (CSI) > 5.
  • HBSS Hanks' balanced salt solution
  • P Neural Tissue Dissociation Kit
  • the tissue was enzymatically digested at 37 °C for 15 min instead of 35 min and the resulting cell suspension was passed through a 40 um cell strainer (Falcon Cell Strainers, Sterile, Corning, product 352340) instead of a MACS SmartStrainer, 70 um.
  • the resulting cell suspension was then stained using allophycocyanin (APC)-conjugated CD1 lb mouse clone Ml/70.15.11.5 (Miltenyi Biotec, 130-098-088) and phycoerythrin (PE)-conjugated CD45 antibody (BD Pharmingen, 553081) according to the manufacturer's (Miltenyi Biotec) recommendations.
  • APC allophycocyanin
  • PE phycoerythrin
  • BD Pharmingen 553081
  • FACS was then used to purify CDllb and CD45 positive microglial cells. Standard, strict side scatter width versus area and forward scatter width versus area criteria were used to discriminate doublets and gate only singlets. Viable cells were identified by staining with propidium iodide (PI) and gating only Pi-negative cells.
  • PI propidium iodide
  • CDllb and CD45 double positive ells were sorted into 1.5ml centrifuge tubes which contains 500 ⁇ of RNA lysis buffer (QIAGEN, catalogue number 74134) with 1% ⁇ -mercaptoethanol (Sigma- Aldrich, catalogue number M6250). RNA was extracted using RNeasy Plus Mini Kit (QIAGEN, catalogue number 74134) according to the manufacturer's protocol. RNA was eluted and then stored at -80 °C until whole transcriptome amplification, library preparation and sequencing.
  • Isolation of neurons Visual cortex was homogenized in 0.5mL ice cold PBS with protease inhibitors and the suspension was centrifuged at 1600 g for 10 minutes. Pellet was resuspended in 5 mL NF-1 hypotonic buffer, incubated for 5 min, and then Dounce-homogenized (pestle A) with 30 strokes. 5mL NF-1 buffer was added to the suspension, washed pestle with 10 ml NF-1 buffer, for a combined total of 20 mL. Collected all in a 50 conical tube and filtered homogenate with 40 ⁇ mesh filter. Pelleted nuclei at 3,000 rpm (1,600 x g) for 15 min.
  • RNA library preparation Extracted total RNA was subject to QC using an Advanced Analytical-fragment Analyzer before library preparation. SMARTer Stranded Total RNA-Seq Kit - Pico Input was used for the P301S neuronal, CK-p25 neuronal, and P301S microglial RNA-seq. SMART- Seq v4 Ultra Low Input RNA Kit was used for CK-p25 microglia specific RNA-seq. Libraries were prepared according to the manufacturer's instructions, and sequenced on the IUumina Nextseq 500 platform at the MIT BioMicro Center.
  • Sample preparation, reduction, alkylation, and tryptic digestion Visual cortex was dissected out and snap frozen in liquid nitrogen and stored in a -80°C freezer until further use. Samples were subsequently homogenized using a plastic hand-held motor driven homogenizer with freshly prepared 8 M urea solution. The concentration of proteins in samples was quantified using a Bio-Rad protein assay. Samples containing 1 mg of protein per 1 ml were prepared, aliquoted and stored at -80°C freezer until further use.
  • Proteins were reduced with 10 mM dithiothreitol (DTT) for 1 h at 56°C, alkylated with 50 mM iodoacetamide for 1 h at room temperature (RT) and diluted to less than 1M urea with 100 mM ammonium acetate at pH 8.9. Proteins were digested using sequencing grade trypsin (Promega; 1 ⁇ g trypsin per 50 ⁇ g protein) overnight at RT. Enzyme activity was quenched by acidification of the samples with acetic acid.
  • DTT dithiothreitol
  • RT room temperature
  • the peptide mixture was desalted and concentrated on a CI 8 Sep-Pak Plus cartridge (Waters) and eluted with 50% acetonitrile, 0.1% formic acid and 0.1% acetic acid. Solvent was evaporated in a SpeedVac vacuum centrifuge. 400 ⁇ g aliquots of each sample were aliquoted and frozen in liquid nitrogen for 5 min, lyophilized and stored at -80°C.
  • TMT labeling TMT labeling and phosphopeptide enrichment: Lyophilized peptides were labeled with TMT-10-plex Mass Tag Labeling Kits (Thermo). For each TMT multiplex, a pooled sample was included consisting of a combination of equal amounts of peptides from WT, non stimulated and 40 Hz entrained mouse model of neurodegeneration, allowing for relative quantification to a normalization channel.
  • Peptide Fractionation The TMT-labeled peptide pellet was fractioned via high-pH reverse phase HPLC. Peptides were resuspended in lOOuL buffer A (lOmM TEAB, pH8) and separated on a 4.6mm x 250 mm 300Extend-C18, 5um column (Agilent) using an 90 minute gradient with buffer B (90% MeCN, lOmM TEAB, pH8) at a flow rate of lml/min. The gradient was as follows: 1-5% B (0-lOmin), 5-35% B (10-70min), 35-70% B (70-80min), 70% B (80- 90min).
  • Fractions were collected over 75 minutes at 1 minute intervals from 10 min to 85 min. The fractions were concatenated into 15 fractions non-contiguously (1+16+31+46+61, 2+17+32+47+62, etc). The fractions then underwent speed- vac (Thermo Scientific Savant) to near dryness.
  • Phosphopeptide enrichment Phosphopeptides were enriched from each of the 15 fractions using the High-Select Fe-NTA phosphopeptide enrichment kit (Thermo) per manufacturer's instructions.
  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS): Peptides were separated by reverse phase HPLC (Thermo Easy nLClOOO) using a precolumn (made in house, 6 cm of 10 ⁇ CI 8) and a self-pack 5 ⁇ tip analytical column (12 cm of 5 ⁇ CI 8, New Objective) over a 140 minutes 1 gradient before nanoelectrospray using a QExactive Plus mass spectrometer (Thermo). Solvent A was 0.1% formic acid and solvent B was 80% MeCN/0.1% formic acid.
  • the gradient conditions were 0-10% B (0-5 min), 10-30% B (5-105 min), 30-40% B (105-119 min), 40-60% B (119-124 min), 60-100% B (124-126 min), 100% B (126-136 min), 100-0% B (136- 138 min), 0% B (138-140 min), and the mass spectrometer was operated in a data-dependent mode.
  • the parameters for the full scan MS were: resolution of 70,000 across 350-2000 m/z, AGC 3e6 and maximum IT 50 ms.
  • the full MS scan was followed by MS/MS for the top 10 precursor ions in each cycle with a NCE of 34 and dynamic exclusion of 30 s.
  • Raw mass spectral data files were searched using Proteome Discoverer (Thermo) and Mascot version 2.4.1 (Matrix Science). Mascot search parameters were: 10 ppm mass tolerance for precursor ions; 15 mmu for fragment ion mass tolerance; 2 missed cleavages of trypsin; fixed modification were carbamidomethylation of cysteine and TMT lOplex modification of lysines and peptide N-termini; variable modifications were methionine oxidation, tyrosine phosphorylation, and serine/threonine phosphorylation.
  • TMT quantification was obtained using Proteome Discoverer and isotopically corrected per manufacturer's instructions and were normalized to the mean of each TMT channel. Only peptides with a Mascot score greater than or equal to 25 and an isolation interference less than or equal to 30 were included in the data analysis.
  • the washed pre column was connected in series with an in-house packed analytical capillary column [50 um ID x 12 cm packed with 5 urn CI 8 beads (YMC gel, ODSAQ, 12 nm, S- 5 um, AQ12S05)] with an integrated electrospray tip (— 1 um orifice).
  • Peptides were eluted using a 140 min (phosphopeptides) or 90 min (total peptide) gradient from 9 to 70% acetonitrile in 0.2 M acetic acid at a flow rate of 0.2 ml/min, with a flow split of -10,000:1, yielding a final electrospray flow rate of -20 nL/min. A total of 15 fractions from each sample were collected.
  • Phosphopeptides were analyzed using a Thermo Q Exactive Hybrid Quadrupole-Orbitrap Plus mass spectrometer with the following settings: spray voltage, 2 kV; no sheath or auxiliary gas flow, heated capillary temperature, 250°C; S-lens radio frequency level of 50%.
  • the Q Exactive was operated in data-dependent acquisition mode.
  • Full-scan MS spectra [mass/charge ratio (m/z), 350 to 2000; resolution, 70,000 at m/z 200] were detected in the Orbitrap analyzer after accumulation of ions at 3e6 target value based on predictive AGC from the previous scan.
  • Mass spectrometry peptide mapping data analysis Raw mass spectral data files were loaded into Proteome Discoverer version 1.4.1.14 (DBversion: 79) (Thermo) and searched against the mouse SwissProt database using Mascot version 2.4 (Matrix Science). TMT reporter quantification was extracted and isotope corrected in Proteome Discoverer. Tandem mass spectra were matched with an initial mass tolerance of 10 ppm on precursor masses and 15 mmu for fragment ions. Cysteine carbamidomethylation, TMT-labeled lysine and protein N-terminal were searched as fixed modifications.
  • Oxidized methionine, and phosphorylation of serine, threonine, and tyrosine were searched as variable modifications.
  • Minimal peptide length was seven amino acids.
  • the data sets were filtered by ion score >20 for all peptides to ensure high confidence in peptide identification and phosphorylation localization and to achieve an (FDR) below 1% for peptides.
  • Phosphopeptide quantification was normalized based on median relative peptide quantification obtained from the crude peptide analysis to correct for slight variations in sample amount among TMT-channels. For each phosphopeptide, relative quantification was represented as a ratio between TMT ion intensities from each analyzed sample and the included normalization channel.
  • Bioinformatics analysis To identify differentially expressed and phosphorylated peptides with significantly regulated ratios, we chose an arbitrary cutoff of ⁇ 20 % difference with an adjusted P value of ⁇ 0.05. The non-regulated background pool consisted of peptides with ratios between 0.8-1.2. Thus, subsequent bioinformatics analyses included peptides with ratios ⁇ 0.8 and >1.2 relative to their normalization channel deemed as downregulated and upregulated, respectively. The name of proteins from protein accession numbers were converted to gene list using Uniprot ID mapping retrieval tool. Protein networks were obtained by using the STRING database (version 10.5). All active interaction sources except text mining were included and to ensure high confidence, a confidence score over 0.9 was required.
  • GO term enrichment analysis was first performed on terms related to biological processes using the STRING (http://string-db.org), TOPPGENE (toppgene.cchmc.org) and Metascape (http://metascape.org) bioinformatics resources, and later was manually filtered to look for commonly present terms from these three resources. GO terms obtained in TOPPGENE was reported. For each individual mouse line (C57BL6/J, CK-p25, P301S tau-tg and 5XFAD), the analysis included gene sets derived from each pool of differentially regulated total peptide or S/T phosphopeptides (up- and downregulated).
  • Novel object recognition in CK-p25 and P301 S tau mice Mice were introduced into the open field arena and the time spent in the center of an arena is calculated. The next day, mice were re-introduced into the same open field box which now contained an additional two familiar objects (novel objects but will be familiar in the next session) and were allowed to explore the objects for 10 minutes. Following this, they were returned to the same arena 10 minutes after the last exploration, with one of the two objects replaced with the new object. Mice behavior was monitored for 7 minutes. Time spent exploring both the familiar and novel objects was recorded using Noldus and computed offline.
  • Seizure susceptibility Mice were injected with picrotoxin (i.p injection) and placed in an open field arena and recorded for 30 minutes using Noldus and also from a side mounted camera. Distance travelled was calculated offline in Noldus, with seizure severity scored manually.
  • MWM is a test for assessing spatial learning. MWM relies on visual cues to navigate from start locations around the perimeter of an open swimming arena to locate a submerged escape platform. Apparatus consisted of a circular pool (122 cm in diameter), filled with a tap water (22°C-24°C) and a non-toxic white paint added to make the solution opaque. An escape platform (10 cm in diameter) with a blunt protruding edge for better grip was submerged 1 inch under the water level. The pool was divided into four equal quadrants labeled N (north), E (east), S (south) and W (west). Mice were introduced to the maze in a randomized order across trials, from the edge, for trials of 60 seconds. The time required to find the hidden platform (latency) was recorded. The probe test was conducted 24h after the last training trial, with the submerged platform removed. All the training and probe test trials were recorded using Noldus.
  • CK-p25, P301S and 5XFAD mice were subjected to GENUS in the morning (until 12pm) and tested in the MWM in the afternoon (3-7pm) which limited behavioral testing time to only 4 hours per day.
  • all AD mice used in MWM performed a comparable number of total training trails.
  • Inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.
  • inventive concepts may be embodied as one or more methods, of which an example has been provided.
  • the acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.
  • the terms “about” and “approximately” can mean within ⁇ 10% of the recited value (inclusive of the recited value itself).
  • “about 100 [units]” can mean within ⁇ 10% of 100 (e.g., from 90 to 110).
  • the terms “about” and “approximately” can mean within ⁇ 5% of the recited value (inclusive of the recited value itself).
  • the terms “about” and “approximately” can mean within ⁇ 1% of the recited value (inclusive of the recited value itself).
  • the terms “about” and “approximately” can be used interchangeably.
  • a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

Abstract

L'invention concerne des dispositifs, des systèmes et des procédés de traitement de la démence ou de la maladie d'Alzheimer chez un sujet en ayant besoin. Selon un exemple, des stimuli visuels chroniques ayant une fréquence d'environ 30 Hz à environ 50 Hz, et plus particulièrement d'environ 40 Hz, sont administrés de manière non invasive au sujet pour entraîner des oscillations gamma dans de multiples régions cérébrales du sujet, comprenant le cortex préfrontal (PFC) et l'hippocampe. Les oscillations gamma entraînées modulent l'activité neuronale à travers de multiples régions cérébrales (par exemple, facilitent la liaison fonctionnelle de réseaux neuronaux à de basses fréquences gamma) pour induire divers effets neuroprotecteurs (par exemple, amélioration des plaques amyloïdes et de l'hyperphosphorylation de Tau) et réduire la neurodégénérescence. L'activité neuronale médiée par les stimuli visuels chroniques réduit une réponse immunitaire de la microglie et améliore les gènes et protéines modifiés de manière aberrante impliqués dans le trafic membranaire, le transport intracellulaire, la fonction synaptique, la neuro-inflammation et la réponse à l'endommagement de l'ADN. Une modification du comportement comprenant un apprentissage et une mémoire améliorés est observée.
PCT/US2018/055258 2017-10-10 2018-10-10 Traitement de la démence par stimulation visuelle pour induire des oscillations gama synchronisées dans le cerveau WO2019075094A1 (fr)

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JP2020520207A JP2020536643A (ja) 2017-10-10 2018-10-10 脳のガンマ振動を同期化する視覚刺激を用いた認知症の治療
CN201880077874.3A CN111655319A (zh) 2017-10-10 2018-10-10 用视觉刺激同步脑中γ振荡治疗痴呆
EP18866752.1A EP3694593A4 (fr) 2017-10-10 2018-10-10 Traitement de la démence par stimulation visuelle pour induire des oscillations gama synchronisées dans le cerveau
KR1020207013291A KR20200086277A (ko) 2017-10-10 2018-10-10 뇌에서 감마 진동을 동기화하기 위해 시각 자극을 이용한 치매 치료
PCT/US2018/055258 WO2019075094A1 (fr) 2017-10-10 2018-10-10 Traitement de la démence par stimulation visuelle pour induire des oscillations gama synchronisées dans le cerveau
CA3078739A CA3078739A1 (fr) 2017-10-10 2018-10-10 Traitement de la demence par stimulation visuelle pour induire des oscillations gama synchronisees dans le cerveau
KR1020237001501A KR20230015501A (ko) 2017-10-10 2018-10-10 뇌에서 감마 진동을 동기화하기 위해 시각 자극을 이용한 치매 치료
AU2018347366A AU2018347366B2 (en) 2017-10-10 2018-10-10 Treating dementia with visual stimulation to synch gamma oscillations in brain
AU2022271389A AU2022271389A1 (en) 2017-10-10 2022-11-15 Treating dementia with visual stimulation to synch gamma oscillations in brain

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PCT/US2018/051785 WO2019074637A1 (fr) 2017-10-10 2018-09-19 Systèmes et procédés de prévention, atténuation et/ou traitement de la démence
USPCT/US2018/051785 2018-09-19
PCT/US2018/055258 WO2019075094A1 (fr) 2017-10-10 2018-10-10 Traitement de la démence par stimulation visuelle pour induire des oscillations gama synchronisées dans le cerveau

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KR102425066B1 (ko) * 2022-03-25 2022-07-28 (주)리솔 실시간 eeg 신호 모니터링을 기초로, 뇌에서 동기화된 뇌파진동을 동조시키는 맞춤형 경두개교류전기자극 장치 및 방법
KR102577515B1 (ko) 2022-11-21 2023-09-12 이종설 파동자극 생성기기

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