Recombinant Mouse PR domain zinc finger protein 13 (Prdm13)

What is the molecular structure and classification of Prdm13?

Prdm13 (PR domain containing protein 13) belongs to the PRDM family of transcriptional regulators, characterized by a positive regulatory (PR) domain and a variable number of zinc finger domains. PRDM factors modulate transcriptional activity either through direct histone methyltransferase activity via their PR domains or by recruiting other histone-modifying enzymes to chromatin . The human PRDM13 gene is located on chromosome 6, specifically at chromosomal band q16.2, with genomic reference NC_000006.11 and transcript reference NM_021620.3 .

What are the primary neurological functions of Prdm13?

Prdm13 serves multiple crucial functions in the central nervous system:

• Controls GABAergic fate in the spinal cord and retina

• Regulates neurogenesis in the hypothalamus

• Modulates sleep-wake patterns through signaling in the dorsomedial hypothalamus (DMH)

• Influences cerebellar development by regulating PAX2+ progenitors

• Affects reproductive neuroendocrine function through regulation of Kiss1 neurons

• Controls metabolic homeostasis, with deficiency leading to increased adiposity

How is Prdm13 expression regulated in the hypothalamus?

In the hypothalamus, particularly in the compact region of the dorsomedial hypothalamus (DMC), Prdm13 expression is regulated by several factors:

• Transcriptionally activated by Nk2 homeobox 1 (Nkx2-1), with knockdown of Nkx2-1 suppressing Prdm13 expression in primary hypothalamic neurons

• Expression increases under dietary restriction conditions

• Shows diurnal oscillation patterns

• Decreases significantly with advancing age

• Is upregulated in long-lived brain-specific Sirt1-overexpressing transgenic (BRASTO) mice

What techniques are most effective for visualizing Prdm13+ cells?

Several complementary techniques have proven effective for Prdm13 detection:

• Genetic labeling using Prdm13-CreERT2 mice crossed with Cre-dependent ZsGreen reporter mice to visualize Prdm13+ cells in different brain regions

• In situ hybridization to confirm colocalization of reporter proteins with endogenous Prdm13 mRNA

• RNAscope analysis for highly sensitive detection of cFos expression in Prdm13+ DMH neurons during experimental manipulations

• Whole-cell patch-clamp techniques to investigate electrophysiological characteristics of Prdm13+ DMH cells

• qRT-PCR to quantify expression levels under different experimental conditions

What phenotypes are observed in Prdm13-deficient mouse models?

Prdm13-deficient mice exhibit multiple phenotypes depending on the extent and localization of the deficiency:

• Complete null mutations are perinatally lethal, indicating essential developmental functions

• DMH-specific Prdm13 knockdown mice display:

  • Sleep fragmentation and compromised sleep quality

  • Excessive sleepiness during sleep deprivation

  • Progressive increases in body weight and adiposity

  • Decreased physical activity and shortened lifespan

  • Reduced wakefulness during the dark (active) period

• Molecular and cellular alterations include:

  • Marked reduction of Kiss1 mRNA levels in the hypothalamus

  • Decreased Gad1 expression, indicating reduced GABAergic neurotransmission

  • Significant reduction in PAX2+ progenitors in the cerebellar ventricular zone

  • Ectopic expression of glutamatergic lineage markers

What human conditions are associated with PRDM13 mutations?

Recessive mutations in PRDM13 have been associated with a syndrome characterized by:

• Intellectual disability

• Ataxia with cerebellar hypoplasia

• Scoliosis

• Delayed puberty with congenital hypogonadotropic hypogonadism (CHH)

This condition is sometimes referred to as CDIDHH or PCH17 in clinical databases . Human mutations are likely partial loss-of-function, as complete loss-of-function mutations in mice are perinatally lethal .

How does Prdm13 expression change with aging?

Prdm13 expression in the hypothalamus decreases significantly with advancing age . This age-associated decline in Prdm13/Nkx2-1-mediated signaling in the DMC leads to decreased sleep quality and increased adiposity . Notably, Prdm13+ neurons in the DMH are activated during sleep deprivation in young mice but not in old mice , suggesting an age-related functional deterioration that could contribute to age-associated pathophysiology in mammals.

What mechanisms underlie Prdm13's role in sleep-wake regulation?

Prdm13 signaling in the DMH regulates sleep-wake patterns through several mechanisms:

• Prdm13+ neurons in the DMH are activated during sleep deprivation in young mice, suggesting their involvement in promoting wakefulness or countering sleep pressure

• Chemogenetic inhibition of Prdm13+ neurons in the DMH promotes increased sleep attempts during sleep deprivation, confirming their role in maintaining wakefulness under sleep pressure

• DMH-specific Prdm13-knockout mice display:

  • Sleep fragmentation similar to aged mice

  • Increased sleep attempts during sleep deprivation

  • Significantly lower wakefulness during the dark period

The diurnal oscillation pattern of Prdm13 expression suggests it may be under circadian control, potentially linking sleep regulation to circadian rhythms .

How does dietary restriction influence Prdm13 expression and sleep phenotypes?

Dietary restriction (DR) has significant effects on Prdm13 expression and function:

• Prdm13 expression in the hypothalamus increases under dietary restriction conditions

• DR ameliorates age-associated sleep fragmentation and increased sleep attempts during sleep deprivation

• These beneficial effects of DR are abrogated in DMH-Prdm13-KO mice, indicating that Prdm13 signaling is necessary for DR's positive effects on sleep quality

• Mechanistically, DR may enhance Prdm13 expression through Sirt1 signaling, as Prdm13 has been shown to be regulated by Sirt1 in the DMH

This suggests that the DR-Sirt1-Prdm13 axis represents an important pathway mediating the beneficial effects of dietary interventions on sleep quality during aging.

How can chemogenetic approaches be used to manipulate Prdm13+ neurons?

Chemogenetic approaches offer powerful tools for manipulating Prdm13+ neurons:

• Using Designer Receptors Exclusively Activated by Designer Drugs (DREADDs):

  • Express inhibitory DREADD (hM4Di) in Prdm13+ neurons using Prdm13-CreERT2 mouse lines

  • Deliver Cre-dependent AAV vectors carrying DREADD constructs (e.g., AAV-DIO-hM4Di-mCherry) via stereotactic injection into the DMH

  • Administer CNO to temporarily inhibit Prdm13+ neuronal activity

• Experimental validation:

  • hM4Di-expressing Prdm13CreERT2/+ mice showed significantly more sleep attempts for 2 hours after CNO injection during sleep deprivation testing

  • Control mice showed no difference between CNO and vehicle treatment

This approach allows for temporally controlled manipulation of neuronal activity, enabling researchers to distinguish between developmental effects and acute effects of Prdm13+ neuron inhibition.

What are the methodological considerations for DMH-specific Prdm13 knockdown models?

Creating and utilizing DMH-specific Prdm13 knockdown models requires several methodological considerations:

• Knockdown approach:

  • Stereotactic injection of lentiviruses carrying Prdm13 shRNA into the DMH

  • Requires precise stereotactic coordinates targeting the compact region of the DMH

  • Control injections with non-targeting shRNA are essential

• Verification methods:

  • qRT-PCR to confirm reduced expression

  • In situ hybridization to verify spatial specificity

  • Immunohistochemistry if suitable antibodies are available

• Functional assessment:

  • Electroencephalogram (EEG) recording to assess sleep-wake architecture and sleep quality

  • Behavioral tests to assess physical activity and metabolic parameters

  • Challenge paradigms like sleep deprivation to reveal phenotypes

• Considerations:

  • Developmental versus acute effects (complete null mutations are lethal)

  • Age as a critical variable, as phenotypes may be more pronounced in older animals

  • Sex differences in sleep regulation and Prdm13 function

What molecular mechanisms underlie Prdm13's role in neuronal cell fate determination?

Prdm13 functions as an essential GABAergic cell-fate determinant through specific molecular mechanisms:

• Functions downstream of transcription factor PTF1A to promote GABAergic and suppress glutamatergic fate

• Modulates transcriptional activity through direct histone methyltransferase activity or by recruiting histone-modifying enzymes to chromatin

• Regulates Gad1 expression, encoding glutamic acid decarboxylase (key enzyme in GABA synthesis)

• Controls Kiss1 mRNA levels in the hypothalamus, affecting the development or function of Kiss1 neurons

In Prdm13-deficient mice, ectopic expression of the glutamatergic lineage marker TLX3 occurs in the cerebellar ventricular zone, confirming its role in suppressing glutamatergic fate .

How does Prdm13 interact with other transcription factors?

Prdm13 interacts with various transcription factors in complex regulatory networks:

• Nkx2-1 interaction:

  • Nkx2-1 upregulates Prdm13 promoter activity

  • Knockdown of Nkx2-1 suppresses Prdm13 expression in primary hypothalamic neurons

  • Suggests Nkx2-1 acts upstream of Prdm13 in hypothalamic regulatory networks

• PTF1A interaction:

  • Prdm13 functions downstream of PTF1A in the spinal cord and possibly other CNS regions

  • This pathway promotes GABAergic and suppresses glutamatergic fate

  • PTF1A is essential for GABAergic neurogenesis, with Prdm13 as a key effector

These relationships position Prdm13 as an integrator of various transcriptional signals controlling neuronal cell fate decisions and physiological functions.

What are the potential translational applications of Prdm13 research?

Understanding Prdm13 function could lead to several translational applications:

• Age-related sleep disorders:

  • The role of Prdm13 in sleep regulation suggests potential therapeutic targets for age-associated sleep disturbances

  • Strategies to maintain or restore Prdm13 signaling might improve sleep quality in aging populations

• Metabolic disorders:

  • The link between Prdm13, adiposity, and physical activity suggests relevance to obesity and metabolic syndrome

  • Understanding how dietary interventions regulate Prdm13 could inform nutritional approaches

• Reproductive disorders:

  • The connection between Prdm13 and Kiss1 neurons suggests applications for hypogonadotropic hypogonadism and delayed puberty

  • Targeting this pathway might offer new treatment options for reproductive endocrine disorders

• Cerebellar development disorders:

  • The role of Prdm13 in cerebellar development suggests relevance to ataxias and other movement disorders

  • Understanding these mechanisms could inform therapies for cerebellar hypoplasia

What are key unanswered questions in Prdm13 research?

Several important questions remain unanswered:

• What are the specific downstream targets of Prdm13 in different neuronal populations?

• How does Prdm13 integrate signals from metabolic, circadian, and sleep homeostatic pathways?

• What epigenetic mechanisms mediate the age-related decline in Prdm13 expression?

• Can pharmacological interventions targeting the Prdm13 pathway improve age-related sleep and metabolic disorders?

• What is the evolutionary significance of Prdm13's dual role in sleep regulation and reproductive control?

• How does Prdm13 interact with other hypothalamic circuits regulating energy balance and food intake?